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Impaired Expression of the Cell Cycle Regulator BTG2 Is Common in Clear Cell Renal Cell Carcinoma

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Impaired Expression of the Cell Cycle Regulator BTG2 Is Common in Clear Cell Renal Cell Carcinoma [CANCER RESEARCH 64, 1632–1638, March 1, 2004] Impaired Expression of the Cell Cycle Regulator BTG2 Is Common in Clear Cell Renal Cell Carcinoma Kirsten Struckmann, Peter Schraml, Ronald Simon, Katja Elmenhorst, Martina Mirlacher, Juha Kononen, and Holger Moch Institute for Pathology, University of Basel, Basel, Switzerland ABSTRACT carcinoma-1 locus at 3p12, which all have been shown to play a role in the biology of cRCC (12–15). In contrast to loss of 3p, which is The prognosis of patients with renal cell carcinoma (RCC) is poor. A associated with initiation of cRCC (10, 11), loss of 9p and 14q have full understanding of the molecular genetics and signaling pathways been shown to be linked to progression of cRCC (9, 16, 17). Addi- involved in renal cancer development and in the metastatic process is of central importance for developing innovative and novel treatment options. tional cytogenetic alterations in cRCC are losses of 4q, 6q, 13q, and In this study, BD Atlas Human Cancer 1.2 cDNA microarrays were used Xq and gains of 5q, 17p, and 17q (9, 11), suggesting many still to identify genes involved in renal tumorigenesis. By analyzing gene unknown genes involved in the initiation and progression of cRCC. expression patterns of four clear cell RCC (cRCC) cell lines and normal The development of microarray technology platforms allows rapid renal tissue, 25 genes were found differentially expressed. To determine screening and evaluation of molecular markers and signaling path- the relevance of these genes, RNA in situ hybridization was performed on ways important in human cancer. Using cDNA microarrays, the a tissue microarray generated from 61 snap-frozen primary renal cell expression levels of thousands of genes could be assessed in a limited carcinomas and 12 normal renal cortex biopsies. B-cell translocation gene number of samples enabling molecular classification of cancer and the 2 (BTG2), a negative cell cycle regulator, which was expressed in normal identification of molecular signatures that might facilitate prediction renal tissue but down-regulated in cRCC cell lines and primary cRCCs, of disease outcome and response to treatment (18–20). Tissue mi- was selected for additional experiments. Quantitative BTG2 mRNA ex- pression analysis in 42 primary cRCCs and 18 normal renal cortex croarrays (TMAs) have been designed to analyze simultaneously new biopsies revealed up to 44-fold reduced expression in the tumor tissues. cancer-related genes in hundreds of tumors on the DNA, RNA, and Decrease of BTG2 expression was not associated with tumor stage, grade, protein level (21, 22). Consequently, a combination of cDNA mi- and survival. Cell culture experiments demonstrated that BTG2 expres- croarray and TMA technology is particularly suitable for rapid iden- sion was weakly inducible by the phorbolester 12-O-tetradecanoylphor- tification and subsequent validation of potential novel cancer markers bol-13-acetate in one of four cRCC cell lines. In contrast, increasing cell and prognostic parameters. density led to elevated BTG2 mRNA expression in three of four cRCC cell In this study, we used a combination of cDNA microarray analysis lines. In both experiments, BTG2 mRNA levels did not reach values and RNA in situ hybridization (RISH) on TMAs made from snap- observed in normal renal tissue. These data suggest that down-regulation frozen tissue specimens to identify tumor-relevant genes for cRCC. of BTG2 is an important step in renal cancer development. The B-cell translocation gene 2 (BTG2), coding a negative cell cycle regulator, was further analyzed in fresh frozen primary cRCCs and INTRODUCTION normal renal cortex biopsies by quantitative reverse transcription- Renal cell cancer (RCC) accounts for ϳ2% of all human cancers PCR (RT-PCR). Additionally, the regulation of the BTG2 gene was worldwide with an incidence of 189.000 and a mortality of 91,000 in studied in cRCC cell lines. the year 2000 (1). RCC is characterized by absence of early warning signs leading to a rather high percentage of advanced, already meta- static tumors at first presentation. Additionally, 40% of nonmetastatic MATERIALS AND METHODS tumors will become metastatic during the course of disease (2). To Cell Cultures. Human cRCC cell lines Caki-1, Caki-2, 786-O, and 769-P date, there is no known cure for metastatic RCC because those tumors and the human cervix carcinoma cell line HeLa were obtained from American are unresponsive to conventional systemic therapies (3, 4). The 5-year Type Culture Collection. All cell lines were grown in Optimem (Invitrogen, survival rate of patients suffering from metastatic RCC is Ͻ10% (5). San Diego, CA), which was supplemented with 10% FCS (Amimed, Basel, Histopathological tumor stage and grade are well-established prog- Switzerland) and 1% penicillin/streptomycin (Amimed). nostic markers for RCC (6). However, the clinical behavior of RCC is Primary Tumors and Normal Renal Tissues. All primary renal tumors variable and often unpredictable. Identification of new molecular and normal renal tissues used for the TMA construction and the quantitative markers would facilitate outcome predictions and improve therapeutic RT-PCR experiments were taken from our frozen tissue archives. Normal options. For this reason, many efforts have been made to understand tissue samples stemmed exclusively from the renal cortex parenchyma, which predominantly consists of proximal tubules (23). Tumor stage and histological the genetic background of initiation and progression of RCC. Com- subtype were defined according to the recommendations of the Union Inter- plete or partial loss of 3p is linked to clear cell RCC (cRCC)—the national Contre Cancer (24) and the recent RCC classification (7). Histological ϳ most common subtype of RCC accounting for 75% of all RCCs grading was done according to a three-tiered grading system (25). H&E- (7)—and is the most frequent alteration in this renal tumor subtype stained sections were prepared from all tissue samples and were reviewed by (8–11). The short arm of chromosome 3 harbors several potential one pathologist (H. M) to ensure the integrity of the tissue. Representative tumor suppressor genes, including the von Hippel-Lindau gene at tissue areas were marked and used for TMA construction or RNA extraction, 3p25, the RASSF1A gene at 3p21.3, and the nonpapillary renal respectively. RNA Extraction. Total RNA from cRCC cell lines, primary cRCCs, and normal renal cortex biopsies was extracted with TRIzol reagent (Invitrogen) Received 6/10/03; revised 12/22/03; accepted 12/31/03. Grant support: Swiss National Science Foundation Grant 31-63923.00. according to the instructions of the manufacturer. DNase treatment of total The costs of publication of this article were defrayed in part by the payment of page RNA was done using DNase I system in combination with the RNeasy kit charges. This article must therefore be hereby marked advertisement in accordance with (Qiagen, Hilden, Germany). RNA concentrations were determined with a 18 U.S.C. Section 1734 solely to indicate this fact. spectrophotometer. Requests for reprints: Dr. Holger Moch, Institute for Pathology, Schoenbeinstrasse 40, CH-4031 Basel, Switzerland. Phone: 41-61-265-2980; Fax: 41-61-265-3194; E-mail: cDNA Microarray Analysis. Gene expression patterns of cRCC cell lines [email protected]. Caki-1, Caki-2, 786-O, and 769-P and normal renal tissue (Invitrogen) were 1632 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. IMPAIRED BTG2 EXPRESSION IN cRCC Fig. 1. The frozen renal tissue microarray. A, overview of the renal tissue microarray (TMA). B, composition of the renal TMA; cRCC, clear cell renal cell carcinoma; pRCC, papillary RCC; chRCC, chromophobe RCC. C, H&E-stained frozen section of the renal TMA. analyzed using BD Atlas Human Cancer 1.2 cDNA microarrays (BD Bio- Air-dried TMA sections were hybridized with 2–4 ng of radiolabeled probe sciences Clontech, Palo Alto, CA). For each experiment, 5 ␮g of total RNA in hybridization mix (50% formamide, 10% dextransulfate, 2ϫ SSC) in a were used for single-stranded cDNA synthesis using the Atlas Pure Total RNA moist chamber at 42°C overnight. After hybridization, slides were washed in ␣ 32 ϫ ϫ Labeling System (BD Biosciences Clontech) and ( - P) dATP (Amersham 1 SSC at 55°C(4 15 min), incubated in distilled H2O, 60 and 90% ethanol Biosciences, Buckinghamshire, United Kingdom) as a label. Unincorporated (30 s each), and air-dried. Slides were exposed to high-resolution screens for nucleotides were removed using the QIAquick Nucleotide Removal kit (Qia- 48 h (Packard Bioscience Company) prior scanning. gen). Prehybridization, hybridization, and washing of the cDNA microarrays ArrayVision software package (Imaging Research Inc., St. Catharines, were done according to standard protocols. Arrays were exposed to a high- Ontario, Canada) was used for image analysis. In brief, for each experiment resolution screen (Packard Bioscience Company, Toronto, Ontario, Canada) signal density (sDens) values were obtained for all tissue spots on the TMA as for 24 h and scanned (Cyclone; Packard Bioscience Company). AtlasImage well as for the background (measured between tissue spots of a given slide and 1.01a software (BD Biosciences Clontech) was used for digital image analysis. averaged). The mean background sDens values were nearly identical for all Background-corrected signal density (sDens) values were calculated for each slides. To safely distinguish between
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