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Molecular genetic alterations and gene expression profile of a malignant rhabdoid tumor of the kidney
Cancer Genetics and Cytogenetics 163 (2005) 130–137
Molecular genetic alterations and gene expression profile of a malignant rhabdoid tumor of the kidney Toshihito Nagataa,*, Yasuo Takahashia, Yukimoto Ishiia, Satoshi Asaia, Megumi Sugahara-Kobayashia, Yayoi Nishidaa, Akiko Murataa, Shunji Yamamorib, Yoshiyasu Ogawab, Takeshi Nakamurab, Hitohiko Murakamic, Masanori Nakamurac, Hiroyuki Shichinoc, Motoaki Chinc, Kiminobu Sugitod, Taro Ikedad, Tsugumichi Koshinagad, Hideo Mugishimaa a
Department of Advanced Medicine, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan b Genetic Research Laboratory, Mitsubishi Kagaku Bio-clinical Laboratories Inc., 3-30-1 Shimura, Itabashi-ku, Tokyo 174-8555, Japan c Department of Pediatrics, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan d Department of Pediatric Surgery, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan Received 15 March 2005; received in revised form 10 May 2005; accepted 16 May 2005
Abstract
Malignant rhabdoid tumor of the kidney (MRTK) is a rare but highly aggressive tumor in children, and knowledge about the molecular signature of this tumor is limited. We report the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl. Fluorescence in situ hybridization and Southern blot analyses revealed a homozygous deletion of an ~0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. This deleted region encodes SMARCB1, a candidate tumor suppressor gene for MRTK. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with a noncancerous kidney (NK) sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case may have contributed to the biological characteristics of the MRTK tumor that arose in our patient. Ó 2005 Elsevier Inc. All rights reserved.
1. Introduction Malignant rhabdoid tumors (MRTs) are rare but highly aggressive malignancies that arise predominantly in children less than 2 years of age [1–3]. This group of tumors occurs in various anatomic sites, including the kidney, central nervous system (CNS), and soft tissue [1,3,4]. The renal counterpart of MRT is malignant rhabdoid tumor of the kidney (MRTK), which has clinicopathological features distinct from Wilms’ tumor [4,5]. Nearly 15% of MRTKs are associated with the occurrence of nonrhabdoid tumors
* Corresponding author. Tel.: 181-3-3972-8111 ext. 2772; fax: 1813-5917-4670. E-mail address: [email protected] (T. Nagata). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.05.009
of the CNS, such as medulloblastoma and primitive neuroectodermal tumor, with no temporal relationship between them [1,6–8]. MRTK frequently demonstrates widespread metastasis at the time of diagnosis, and the prognosis of patients with this disease remains extremely poor, despite intensive chemotherapy [1,4,9]. The cause of MRTK is largely unknown. The histogenetic origin of this tumor has not yet been determined [3,10]. Most MRTKs demonstrate a normal karyotype, but there have been a few reports of numerical or structural chromosomal abnormalities, involving mainly chromosome 22 [11,12]. Molecular genetic investigations of MRT cells have revealed heterozygous or homozygous deletions at chromosomal locus 22q11.2 [13–20]. Recently, within this region, the SMARCB1 (hSNF5/INI1) gene was isolated as
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a candidate tumor suppressor gene for highly frequent, biallelic inactivating alterations in MRTKs and extrarenal MRTs, leading to tumorigenesis [21–23]. Although genetic alteration of chromosomal region 22q11.2 in MRTK has been reported in a limited number of cases, other molecular bases for the biological characteristics of this tumor, including the gene expression profile, have not yet been clarified [7,8,10,13,15,16,18,19,21,22]. Thus, accumulation of detailed investigations of molecular genetic alterations and the gene expression profile of MRTK tumors may facilitate the identification of candidate genes affecting the biological characteristics of MRTK, and may provide deeper insights into the biological characteristics of this type of tumor. Here, we describe a homozygous deletion of an ~0.29-Mb genomic region at 22q11.23 and the gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl.
2. Materials and methods 2.1. Patient and samples MRTK arising from the right kidney was diagnosed in a 4-month-old Japanese girl, according to the histological classification reported previously [24]. Her family history was unremarkable. Magnetic resonance imaging of the brain also demonstrated a solid and cystic mass in the left cerebellum. The patient underwent right nephrectomy including the tumor and sampling of abdominal lymph nodes adherent to the tumor as well as para-aortic lymph nodes. Histological examination of para-aortic lymph nodes showed metastasis of tumor cells. The patient received chemotherapy with carboplatin, etoposide, and cyclophosphamide, and external beam radiotherapy to the tumor bed at a dose of 1,080 cGy, according to the regimen RTK of the National Wilms’ Tumor Study 5 [25]. Despite intensive chemotherapy, the patient developed progression of the abdominal tumor and multiple metastases in the liver, and died 8 months after diagnosis. Because of the patient’s poor general condition, the cerebellar tumor was neither biopsied nor resected during the clinical course. Samples of the MRTK tumor and noncancerous kidney (NK) were obtained at surgery for the primary tumor before chemotherapy. Half of the tumor sample and NK sample was fixed in 10% formalin and embedded in paraffin for light microscopy, and the remaining half of each sample was frozen at 280 C for subsequent genetic investigations. All samples were acquired after obtaining informed consent from the parents. As normal controls for fluorescence in situ hybridization (FISH) and Southern blot analyses of the tumor, peripheral blood lymphocytes or mononuclear cells were acquired from healthy volunteers after obtaining informed consent. 2.2. Slide preparation and FISH analysis Snap-frozen tissue from the tumor was used to make touch preparations for interphase FISH analysis.
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Preparations of metaphase spreads and interphase cell nuclei of peripheral blood lymphocytes acquired from healthy volunteers were made according to a standard method as described elsewhere [26]. The slides were prepared according to the standard technique. For FISH analysis of the chromosomal region at 22q11.23, four bacterial artificial chromosome (BAC) clones (RP11-61N10, RP11-698L6, RP11-80O7, RP11-297B9) lying within 22q11.23 and a BAC clone RP11-423E19 located at 22q13.33 were selected using information in the Ensembl Genome Database (http://www.ensembl.org/, accessed November, 2004), the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (http://bacpac.chori.org/home.htm), and the UCSC genome browser (http://genome.ucsc.edu/). These BACs were purchased from Invitrogen Life Technologies Co. (Carlsbad, CA). The human genomic DNA fragments derived from BACs located at 22q11.23 were labeled with SpectrumOrange-dUTP, and the human genomic DNA fragment derived from BAC clone RP11-423E19 was labeled with SpectrumGreen-dUTP, using a nick translation kit (Vysis, Downers Grove, IL). After hybridization of aliquots of mixed probes (RP11-423E19 and a probe for 22q11.23) with the prepared slides, the metaphase spreads and interphase cell nuclei were counterstained with 4#,6-diamidino-2-phenylindole and were analyzed using a fluorescence microscope (Microphot-FXA, Nikon, Japan) equipped with a cooled charge-coupled device (CCD) camera (Hamamatsu Photonics, Shizuoka, Japan), as described elsewhere [26]. Using metaphase spreads of peripheral blood lymphocytes, the number and location of hybridization signals of each BAC probe were confirmed, as described elsewhere [26]. For interphase cell nuclei preparations, signals in nonoverlapping, apparently intact nuclei were examined by counting the number of signals for the RP11-423E19 probe and the number of signals detecting the chromosomal region at 22q11.23, as described previously [26]. For all the pairs of BAC probes used, two signals both for the probe detecting chromosomal region 22q11.23 and for the RP11-423E19 probe were observed for O960 out of 1,000 interphase cell nuclei derived from healthy volunteers. 2.3. Genomic DNA analysis Genomic DNA extraction from the MRTK tumor and Southern blot analysis of DNA were performed by standard methods [27]. In brief, 5 mg genomic DNA extracted from the MRTK tumor and peripheral blood mononuclear cells derived from healthy volunteers was digested with restriction enzymes, electrophoresed on 1.0% agarose gel, blotted onto nylon filter membranes, and hybridized with 32Plabeled probes. A probe for the BCR gene, BCR 3# probe (1.2 kb of HindIII–EcoRI genomic DNA fragment in BCR intron 14–15), was purchased (Oncogene Science, Manhasset, NY) [28]. Genomic DNA probes specific for
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the Rgr, SMARCB1, DDT, and ADORA2A genes were generated by amplification of genomic DNA sequences of healthy volunteers using polymerase chain reaction (PCR) (Table 1). Each PCR was performed in a final volume of 20 mL containing 40 ng genomic DNA, 5.0 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) with 10 PCR buffer, and the primer set for the target sequence at a final concentration of 0.5 mmol/L. The authenticity of each PCR product was confirmed by direct sequencing, as described previously [29]. After hybridization, the filter membranes were exposed to Fuji HR-HA film at 280 C using an intensifying screen.
2.4. cRNA preparation and hybridization to oligonucleotide DNA array Total RNA extraction, cRNA preparation, and hybridization of cRNA on the Human Cancer G110 array (G110 Cancer Array), carrying 1992 probe sets representing O1,700 genes that have been implicated in cancer biology, were performed using the standard Affymetrix protocol (Affymetrix, Santa Clara, CA), as described previously [30]. For the present study, we independently prepared three cRNA pools, two derived from the NK sample and one derived from the MRTK tumor sample, and each cRNA
was hybridized on the G110 Cancer Array for the following investigations. 2.5. Microarray data analysis After scaling of each image file to an average hybridization intensity of 200, the expression level (average difference) for each gene was determined by calculating the average difference in intensity (perfect match – mismatch) between its probe pairs using GeneChip Suite 3.3 software (Affymetrix), as described previously [30,31]. We use the term signal intensity here to describe the hybridization intensity of each gene in the GeneChip data. To eliminate unreliable data, genes with a signal intensity of !0 in at least one sample and genes whose signal intensity was regarded as ‘‘absent’’ by the computational algorithm of GeneChip Suite 3.3 software (Affymetrix) across all samples were excluded, as described previously [30,31]. 2.6. Selection of probe sets with reproducibility and those showing alteration of signal intensity To select probe sets with reproducibility of signal intensity in this experiment, we performed pairwise comparison of the signal intensities of probe sets derived from two independently prepared cRNA pools of the NK sample, as
Table 1 Primer sets for genes and conditions used for polymerase chain reaction (PCR) to generate probes for Southern blot analysis Gene symbol
GenBank accession no.
Rgr
AC000347
SMARCB1
AP000349
DDT
AP000351
ADORA2A
AP000355
Gene name
PCR primer seta
Conditions for PCR
forward primer: (94 C 5 min)–(94 C 30 sec, 5#-GCCCATAGCAAAAGCATGAAAG-3# 54 C 30 sec, 72 C 45 sec) 35 cycles–(72 C 5 min) (ch22:22,361,683-22,361,704) reverse primer: 5#-ATACAGTCGTGATGGGAAAGGG-3# (ch22:22,362,768-22,362,789) forward primer: (94 C 5 min)–(94 C 30 sec, SWI/SNF related, 5#-GGTGTCCTGTGTCCTCCAGC-3# matrix associated, 55 C 30 sec, 72 C 30 sec) actin dependent (ch22:22,469,412-22,469,431) 40 cycles–(72 C 5 min) regulator of reverse primer: chromatin, 5#-GAAACGGGACTGTTCCCACG-3# subfamily b, (ch22:22,470,214-22,470,233) member 1 D-dopachrome forward primer: (94 C 5 min)–(94 C 30 sec, 61 C 30 sec, 72 C 30 sec) tautomerase 5#-TCTAATAGGGCTAGGAACCG-3# 35 cycles–(72 C 5 min) (ch22:22,641,496-22,641,515) reverse primer: 5#-AGACCCTGATGTGTCCTTAC-3# (ch22: 22,640,396-22,640,415) Adenosine forward primer: (94 C 5 min)–(94 C 30 sec, A2a receptor 5#-ACCTGCAGAACGTCACCAACT 54 C 30 sec, 72 C 45 sec) ACT-3# (ch22:23,154,033-23,154,056) 35 cycles–(72 C 5 min) reverse primer: 5#-CCATGCCGAGTAATT CAGCCTCT-3# (ch22:23,154,984-23,155,006) Ral-GDS related protein Rgr
Size of PCR product (bp)b 1107
822
1120
974
Abbreviations: min, minutes; sec, seconds. The sequence information described in this table was collected in November, 2004 using the Ensembl Genome Database (http://www.ensembl.org/). a Numbers within parentheses denote the coordinates of each primer on chromosome 22. b Expected size of each PCR product calculated according to Human Genome Sequence Build 35.
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described previously [31]. We selected the probe sets whose differences in signal intensity were less than 2.0-fold in this comparison as probe sets with reproducibility of signal intensity, as described elsewhere [32]. Using the probe sets with reproducibility of signal intensity, we next performed two pairwise comparisons between the data sets of the NK sample (n 5 2) and the MRTK tumor sample (n 5 1). In the present study, to get a picture of the gene expression profile of MRTK, we consequently selected probe sets showing a marked alteration of signal intensity that was more than 4.0-fold higher or lower in the MRTK tumor sample than in the NK sample in all pairwise comparisons, as described previously [31]. All the procedures used for selection of probe sets were performed using GeneSpring 4.2 software (Agilent Technologies, Palo Alto, CA). 2.7. Functional classification of genes Functional classification of genes was performed according to the classification of the Gene Ontology Consortium (http://www.geneontology.org) and iterative PubMed search (National Center for Biotechnology Information– NCBI, Bethesda, MD) [33].
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3. Results 3.1. Molecular genetic alterations of chromosomal region 22q11.23 in MRTK cells To investigate the molecular genetic alterations in MRTK in our patient, we first analyzed the molecular cytogenetic alterations in chromosomal region 22q11.23 in MRTK cells using FISH analysis. Interphase FISH analysis using a BAC probe RP11-698L6 or RP11-80O7 showed the absence of signals for these two probes in O995 out of 1,000 tumor cell nuclei examined, and two signals for RP11-423E19 at 22q13.33 (green signals) were present in all 1,000 tumor cell nuclei examined (Fig. 1). Interphase FISH analysis using a BAC probe RP11-61N10 or RP11297B9 revealed the presence of two signals (orange signals) for each probe in O960 out of 1,000 tumor cell nuclei examined (Fig. 1). To confirm the results obtained by FISH analyses, we investigated the genomic region at 22q11.23 in MRTK cells using Southern blot analysis. As shown in Fig. 2, Southern blot analyses using Rgr, SMARCB1, and DDT probes detected a homozygous deletion of these genes in MRTK cells. Southern blot analyses for BCR and ADORA2A
Fig. 1. Schematic representation of chromosomal deletion region at 22q11.23 in malignant rhabdoid tumor of the kidney (MRTK) cells in our patient. The FISH signal patterns obtained using BAC probes for chromosomal region 22q11.23 (orange signals indicated by arrows) and the RP11-423E19 probe (green signals indicated by arrowheads) are shown with a map of chromosomal region 22q11.23. The position of each BAC within chromosomal region 22q11.23 is denoted as a black bar with an approximate sequence position on chromosome 22 according to the information in Ensembl Genome Database (http://www.ensembl.org/). The broken line indicates an ~0.29-Mb homozygously deleted region found in MRTK cells, that was confirmed by FISH and Southern blot analyses (see Fig. 2). Coordinates of genes and BACs on chromosome 22 are shown in megabases from 22ptel. Abbreviations: MRTK, malignant rhabdoid tumor of the kidney. a Coordinates of known genes located within or around the homozygously deleted region at 22q11.23 found in MRTK cells in our patient. b Positions of BACs in chromosomal region 22q11.23.
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Fig. 2. Southern blot analyses of malignant rhabdoid tumor of the kidney. Numbers on the left side of M show the size marker. (A) Rgr gene. (B) SMARCB1 gene. (C) DDT gene. (D) BCR gene. (E) ADORA2A gene. Capital letters at the top of each lane or after GL refer to the restriction enzymes used: B, BamHI; E, EcoRI; GII, BglII; K, KpnI; S, SacI. Other abbreviations: GL, germline band; M, size marker; MRTK, DNA derived from malignant rhabdoid tumor of the kidney; NC, normal control DNA derived from peripheral blood mononuclear cells of healthy volunteers.
detected neither rearranged bands nor diminished signals for germline bands (Fig. 2). Considering the results obtained by FISH analyses and the sequence information of this genomic region, the results presented here indicate homozygous deletion of an ~0.29-Mb genomic region bordered by Rgr and DDT and the retention of genomic regions detected with BCR and ADORA2A probes for Southern blot analysis, in MRTK cells in our patient (Figs. 1 and 2).
3.2. Genes differentially expressed between NK sample and MRTK tumor sample To investigate the transcriptional profile of MRTK, we analyzed the differences in gene expression profile between the NK sample and MRTK tumor sample. We first selected 1,020 probe sets as those with reproducibility of signal intensity in this experiment, as described in Section 2.6. Among 1,020 probe sets selected, we obtained 25 probe sets corresponding to 25 genes showing more than 4.0-fold higher expression in the MRTK tumor sample than in the NK sample and 64 probe sets corresponding to 64 genes showing more than 4.0-fold lower expression in the MRTK tumor sample than in the NK sample (Table 2). Among the 64 genes showing decreased expression in the MRTK tumor sample, MIF and DDT genes, which are mapped on the homozygously deleted region found in our case and are included in the G110 Cancer Array used in our
experiment, were found to display lower expression in the MRTK tumor sample (Table 2; Figs. 1 and 2). The detailed functional classifications of genes showing increased or decreased expression in the MRTK tumor sample are also summarized in Table 2.
4. Discussion MRTK is a relatively uncommon solid tumor in children, compared with other types of pediatric cancer, and knowledge about the molecular signature of this tumor is limited. We therefore analyzed the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in our patient. Using FISH and Southern blot analyses, we found that an ~0.29-Mb genomic region bordered by the Rgr and DDT genes was homozygously deleted in these MRTK cells (Figs. 1 and 2). Recent reports regarding the aberrant genomic organization of chromosomal region 22q11.23 in MRT cells suggested the existence of a homozygous deletion within the genomic region bordered by sequence-tagged sites (STSs) D22S556 and D22S42 in some cases of MRT, and the SMARCB1 gene, a candidate tumor suppressor gene, located within this region has been found to show frequent, biallelic inactivating alterations in MRT cells [14,15,17,19–22]. As shown in Figs. 1 and 2, this gene was also inactivated by a homozygous deletion in the MRTK in our patient, and this finding supports the notions
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Table 2 Functional classification of 89 genes showing more than 4.0-fold higher (25 genes) or lower (64 genes) expression in MRTK tumor sample than in noncancerous kidney (NK) sample
Biological function
Genes showing increased expression in MRTK (MRTKONK; 25 genes)
Signal transduction
WNT5A, DDR2, MDK, TGFB3, FZD2
Cell cycle
DTYMK, MKI67, ABL1, PCNA, MAD2L1, PRKDC, CCNE1, TK1, TP53, GAS1 IGF2, IGFBP6, EMP1, TFRC, ABL1 IGF2, MKI67, MDK, NAP1L1, NME1
Cell growth Cell proliferation Cell metabolism Cell adhesion
DDR2
Transcription Apoptosis Immune response DNA replication DNA repair Stress response Cell-cell communication DNA metabolism RNA metabolism DNA damage response Unfolded protein binding activity Protein transport Protein phosphatase regulator activity Transporter activity Muscle contraction Unknown fuction
CRABP2, NFIB
PCNA, TOP2A, NAP1L1 PRKDC TGFB3 TK1 HNRPU ABL1
Genes showing decreased expression in MRTK (NKOMRTK; 64 genes) IL1R1, EGFR, PTHR1, MUC1, NR4A1, NR4A2, PLCL1, TACSTD1, FGFR4, GDF15, PLCG2, EDNRA, S100A2, FGF9, FKBP1B, ERBB3, DAB2, FGF1, BCR, PDGFRB, TACSTD2, PTPRK DUSP1, GADD45A, FOS, ACPP PTN, EGFR, PTHR1, IGFBP7, IGFBP2, PDGFRB EDNRA, FGF9, EGFR, ERBB3, DAB2, FGF1 HYAL1, MMP7, DDT, MT1G, GPX3, ENPP1, PLCG2, PCCA, ACP5, GSTM3, ASAH1 TACSTD1, CDH1, ITGA1, CCL2, LAMB1, CX3CL1, ICAM2, PTPRK, NID2 NR4A1, ESRRA, FOS, NR4A2, CEBPD, HOXD4 BAD, TNFSF10, GADD45A, CASP4 MIF, CD24, MX2, IFITM2 GADD45A DUSP1, SGK PTHR1
HSPA2 CAV2 PPP2R3A ABCB1 MYH11 TM4SF1, C5orf18
Abbreviations: NK, noncancerous kidney; MRTK, malignant rhabdoid tumor of the kidney. Genes are described using gene symbols.
suggested by other authors. In addition, according to information on the human genome sequence, several genes and putative transcriptional units other than SMARCB1 have recently been identified within the homozygously deleted region found in our case (Ensembl Genome Database, http:// www.ensembl.org/). Further characterization of this genomic region in more MRTK tumors may provide additional information on genes affecting the biological characteristics of this tumor. Using a high-density oligonucleotide DNA array, we found 25 genes showing more than 4.0-fold higher expression in the MRTK tumor sample and 64 genes showing more than 4.0-fold lower expression in the MRTK tumor sample, than in the NK sample (Table 2). The decreased expression of MIF and DDT found in our case was suspected to be the result of homozygous deletion of a genomic region at 22q11.23 in the MRTK cells during tumorigenesis, although we failed to analyze the status of this chromosomal region in the patient’s noncancerous cells because samples for FISH and DNA analysis were not available (Table 2; Figs. 1 and 2). The 25 genes showing increased expression in the MRTK tumor sample included genes regulating the cell cycle (10 genes: DTYMK, MKI67, ABL1, PCNA, MAD2L1,
PRKDC, CCNE1, TK1, TP53, GAS1), DNA replication (3 genes: PCNA, TOP2A, NAP1L1), and cell proliferation (5 genes: IGF2, MKI67, MDK, NAP1L1, NME1) (Table 2). Among these genes, increased expression of p53 protein in MRTK cells has been reported to be associated with aggressive biological behavior of this tumor [34]. Increased expression of MKI67, PCNA, and NAP1L1 has been proved to be related to progression of cell growth [35–37], suggesting acceleration of cell division and tumor cell growth in this MRTK tumor. The increased expression of genes regulating cell growth (5 genes: IGF2, IGFBP6, EMP1, TFRC, ABL1) in this MRTK tumor sample also suggests alterations of signaling for cell growth in this MRTK tumor [38–41] (Table 2). Thus, the results obtained in the present study suggest that an altered pattern of signaling for tumor cell growth may have contributed to the biological characteristics of the MRTK tumor in our patient and may have led to the activation of tumor cell growth. The 64 genes showing decreased expression in the MRTK tumor sample included genes regulating apoptosis (4 genes: BAD, TNFSF10, GADD45A, CASP4) (Table 2). Among these changes, decreased expression of TNFSF10 in MRTK tumors has been reported previously [42]. Alterations of expression of genes regulating apoptosis in MRTK
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tumors have been reported by other authors, and some of them have been proposed as therapeutic targets for MRTK tumors [42,43]. Thus, our results support the previously described notion and may provide additional information on the expression profile of genes regulating apoptosis in MRTK tumors [42,43]. In the present study, we found that IGF2, IGFBP6, and MDK showed increased expression, whereas IGFBP2, IGFBP7, and PTN showed decreased expression in the MRTK tumor sample (Table 2). Among these genes, IGF2, IGFBP2, IGFBP6, and IGFBP7 are members of the IGF-axis, and MDK and PTN are members of the midkine family of growth and differentiation factors [38,44]. Alteration of the expression of IGF2 and MDK has been shown in some MRTK tumors and/or cell lines, and these changes are considered to affect the biological characteristics of this tumor [10,45–49]. A recent report has also indicated direct control of PTN expression by SMARCB1 protein via transcriptional activation in an MRT cell line [50]. Thus, the altered pattern of expression of some IGFaxis members and members of the midkine family of growth and differentiation factors found in the present study supports the significance of these alterations in the biological characteristics of MRTK. A recent report describing gene expression profiling of pediatric CNS embryonal tumors, including MRTs, has shown that some genes expressed during myogenesis, such as TPM2, CNN2, NFATC4, and MRCL3, were included in the set of genes discriminating MRTs from other pediatric CNS tumors, and the authors therefore suggested a mesenchymal origin for MRTs [51]. We could not find differential expression of such marker genes between the NK sample and MRTK tumor sample, because the probe sets for marker genes described in the previous report were not included in the microarray used in our study (Table 2). Further analyses of the expression profile of more genes in MRTK tumors may provide a clue to understand the histogenetic origin of this tumor. In summary, we analyzed the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in our patient. FISH and Southern blot analyses of chromosomal region 22q11.23 revealed a homozygous deletion of an ~0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with the NK sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case
may have contributed to the biological characteristics of the MRTK tumor that arose in our patient.
Acknowledgments This work was supported in part by a Grant from the Ministry of Education, Culture, Sports, Science, and Technology to promote advanced scientific research to Nihon University, School of Medicine, by Grant-in-Aids from the Ministry of Education, Science, Sports and Culture of Japan (no. 17591119 and 16591359), by a Nihon University Research Grant for Assistants and Young Researchers (no. 05-031), and by a Nihon University Joint Research Grant (no. 05-016).
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Molecular genetic alterations and gene expression profile of a malignant rhabdoid tumor of the kidney Toshihito Nagataa,*, Yasuo Takahashia, Yukimoto Ishiia, Satoshi Asaia, Megumi Sugahara-Kobayashia, Yayoi Nishidaa, Akiko Murataa, Shunji Yamamorib, Yoshiyasu Ogawab, Takeshi Nakamurab, Hitohiko Murakamic, Masanori Nakamurac, Hiroyuki Shichinoc, Motoaki Chinc, Kiminobu Sugitod, Taro Ikedad, Tsugumichi Koshinagad, Hideo Mugishimaa a
Department of Advanced Medicine, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan b Genetic Research Laboratory, Mitsubishi Kagaku Bio-clinical Laboratories Inc., 3-30-1 Shimura, Itabashi-ku, Tokyo 174-8555, Japan c Department of Pediatrics, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan d Department of Pediatric Surgery, Nihon University, School of Medicine, 30-1, Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan Received 15 March 2005; received in revised form 10 May 2005; accepted 16 May 2005
Abstract
Malignant rhabdoid tumor of the kidney (MRTK) is a rare but highly aggressive tumor in children, and knowledge about the molecular signature of this tumor is limited. We report the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl. Fluorescence in situ hybridization and Southern blot analyses revealed a homozygous deletion of an ~0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. This deleted region encodes SMARCB1, a candidate tumor suppressor gene for MRTK. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with a noncancerous kidney (NK) sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case may have contributed to the biological characteristics of the MRTK tumor that arose in our patient. Ó 2005 Elsevier Inc. All rights reserved.
1. Introduction Malignant rhabdoid tumors (MRTs) are rare but highly aggressive malignancies that arise predominantly in children less than 2 years of age [1–3]. This group of tumors occurs in various anatomic sites, including the kidney, central nervous system (CNS), and soft tissue [1,3,4]. The renal counterpart of MRT is malignant rhabdoid tumor of the kidney (MRTK), which has clinicopathological features distinct from Wilms’ tumor [4,5]. Nearly 15% of MRTKs are associated with the occurrence of nonrhabdoid tumors
* Corresponding author. Tel.: 181-3-3972-8111 ext. 2772; fax: 1813-5917-4670. E-mail address: [email protected] (T. Nagata). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.05.009
of the CNS, such as medulloblastoma and primitive neuroectodermal tumor, with no temporal relationship between them [1,6–8]. MRTK frequently demonstrates widespread metastasis at the time of diagnosis, and the prognosis of patients with this disease remains extremely poor, despite intensive chemotherapy [1,4,9]. The cause of MRTK is largely unknown. The histogenetic origin of this tumor has not yet been determined [3,10]. Most MRTKs demonstrate a normal karyotype, but there have been a few reports of numerical or structural chromosomal abnormalities, involving mainly chromosome 22 [11,12]. Molecular genetic investigations of MRT cells have revealed heterozygous or homozygous deletions at chromosomal locus 22q11.2 [13–20]. Recently, within this region, the SMARCB1 (hSNF5/INI1) gene was isolated as
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a candidate tumor suppressor gene for highly frequent, biallelic inactivating alterations in MRTKs and extrarenal MRTs, leading to tumorigenesis [21–23]. Although genetic alteration of chromosomal region 22q11.2 in MRTK has been reported in a limited number of cases, other molecular bases for the biological characteristics of this tumor, including the gene expression profile, have not yet been clarified [7,8,10,13,15,16,18,19,21,22]. Thus, accumulation of detailed investigations of molecular genetic alterations and the gene expression profile of MRTK tumors may facilitate the identification of candidate genes affecting the biological characteristics of MRTK, and may provide deeper insights into the biological characteristics of this type of tumor. Here, we describe a homozygous deletion of an ~0.29-Mb genomic region at 22q11.23 and the gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl.
2. Materials and methods 2.1. Patient and samples MRTK arising from the right kidney was diagnosed in a 4-month-old Japanese girl, according to the histological classification reported previously [24]. Her family history was unremarkable. Magnetic resonance imaging of the brain also demonstrated a solid and cystic mass in the left cerebellum. The patient underwent right nephrectomy including the tumor and sampling of abdominal lymph nodes adherent to the tumor as well as para-aortic lymph nodes. Histological examination of para-aortic lymph nodes showed metastasis of tumor cells. The patient received chemotherapy with carboplatin, etoposide, and cyclophosphamide, and external beam radiotherapy to the tumor bed at a dose of 1,080 cGy, according to the regimen RTK of the National Wilms’ Tumor Study 5 [25]. Despite intensive chemotherapy, the patient developed progression of the abdominal tumor and multiple metastases in the liver, and died 8 months after diagnosis. Because of the patient’s poor general condition, the cerebellar tumor was neither biopsied nor resected during the clinical course. Samples of the MRTK tumor and noncancerous kidney (NK) were obtained at surgery for the primary tumor before chemotherapy. Half of the tumor sample and NK sample was fixed in 10% formalin and embedded in paraffin for light microscopy, and the remaining half of each sample was frozen at 280 C for subsequent genetic investigations. All samples were acquired after obtaining informed consent from the parents. As normal controls for fluorescence in situ hybridization (FISH) and Southern blot analyses of the tumor, peripheral blood lymphocytes or mononuclear cells were acquired from healthy volunteers after obtaining informed consent. 2.2. Slide preparation and FISH analysis Snap-frozen tissue from the tumor was used to make touch preparations for interphase FISH analysis.
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Preparations of metaphase spreads and interphase cell nuclei of peripheral blood lymphocytes acquired from healthy volunteers were made according to a standard method as described elsewhere [26]. The slides were prepared according to the standard technique. For FISH analysis of the chromosomal region at 22q11.23, four bacterial artificial chromosome (BAC) clones (RP11-61N10, RP11-698L6, RP11-80O7, RP11-297B9) lying within 22q11.23 and a BAC clone RP11-423E19 located at 22q13.33 were selected using information in the Ensembl Genome Database (http://www.ensembl.org/, accessed November, 2004), the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (http://bacpac.chori.org/home.htm), and the UCSC genome browser (http://genome.ucsc.edu/). These BACs were purchased from Invitrogen Life Technologies Co. (Carlsbad, CA). The human genomic DNA fragments derived from BACs located at 22q11.23 were labeled with SpectrumOrange-dUTP, and the human genomic DNA fragment derived from BAC clone RP11-423E19 was labeled with SpectrumGreen-dUTP, using a nick translation kit (Vysis, Downers Grove, IL). After hybridization of aliquots of mixed probes (RP11-423E19 and a probe for 22q11.23) with the prepared slides, the metaphase spreads and interphase cell nuclei were counterstained with 4#,6-diamidino-2-phenylindole and were analyzed using a fluorescence microscope (Microphot-FXA, Nikon, Japan) equipped with a cooled charge-coupled device (CCD) camera (Hamamatsu Photonics, Shizuoka, Japan), as described elsewhere [26]. Using metaphase spreads of peripheral blood lymphocytes, the number and location of hybridization signals of each BAC probe were confirmed, as described elsewhere [26]. For interphase cell nuclei preparations, signals in nonoverlapping, apparently intact nuclei were examined by counting the number of signals for the RP11-423E19 probe and the number of signals detecting the chromosomal region at 22q11.23, as described previously [26]. For all the pairs of BAC probes used, two signals both for the probe detecting chromosomal region 22q11.23 and for the RP11-423E19 probe were observed for O960 out of 1,000 interphase cell nuclei derived from healthy volunteers. 2.3. Genomic DNA analysis Genomic DNA extraction from the MRTK tumor and Southern blot analysis of DNA were performed by standard methods [27]. In brief, 5 mg genomic DNA extracted from the MRTK tumor and peripheral blood mononuclear cells derived from healthy volunteers was digested with restriction enzymes, electrophoresed on 1.0% agarose gel, blotted onto nylon filter membranes, and hybridized with 32Plabeled probes. A probe for the BCR gene, BCR 3# probe (1.2 kb of HindIII–EcoRI genomic DNA fragment in BCR intron 14–15), was purchased (Oncogene Science, Manhasset, NY) [28]. Genomic DNA probes specific for
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the Rgr, SMARCB1, DDT, and ADORA2A genes were generated by amplification of genomic DNA sequences of healthy volunteers using polymerase chain reaction (PCR) (Table 1). Each PCR was performed in a final volume of 20 mL containing 40 ng genomic DNA, 5.0 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) with 10 PCR buffer, and the primer set for the target sequence at a final concentration of 0.5 mmol/L. The authenticity of each PCR product was confirmed by direct sequencing, as described previously [29]. After hybridization, the filter membranes were exposed to Fuji HR-HA film at 280 C using an intensifying screen.
2.4. cRNA preparation and hybridization to oligonucleotide DNA array Total RNA extraction, cRNA preparation, and hybridization of cRNA on the Human Cancer G110 array (G110 Cancer Array), carrying 1992 probe sets representing O1,700 genes that have been implicated in cancer biology, were performed using the standard Affymetrix protocol (Affymetrix, Santa Clara, CA), as described previously [30]. For the present study, we independently prepared three cRNA pools, two derived from the NK sample and one derived from the MRTK tumor sample, and each cRNA
was hybridized on the G110 Cancer Array for the following investigations. 2.5. Microarray data analysis After scaling of each image file to an average hybridization intensity of 200, the expression level (average difference) for each gene was determined by calculating the average difference in intensity (perfect match – mismatch) between its probe pairs using GeneChip Suite 3.3 software (Affymetrix), as described previously [30,31]. We use the term signal intensity here to describe the hybridization intensity of each gene in the GeneChip data. To eliminate unreliable data, genes with a signal intensity of !0 in at least one sample and genes whose signal intensity was regarded as ‘‘absent’’ by the computational algorithm of GeneChip Suite 3.3 software (Affymetrix) across all samples were excluded, as described previously [30,31]. 2.6. Selection of probe sets with reproducibility and those showing alteration of signal intensity To select probe sets with reproducibility of signal intensity in this experiment, we performed pairwise comparison of the signal intensities of probe sets derived from two independently prepared cRNA pools of the NK sample, as
Table 1 Primer sets for genes and conditions used for polymerase chain reaction (PCR) to generate probes for Southern blot analysis Gene symbol
GenBank accession no.
Rgr
AC000347
SMARCB1
AP000349
DDT
AP000351
ADORA2A
AP000355
Gene name
PCR primer seta
Conditions for PCR
forward primer: (94 C 5 min)–(94 C 30 sec, 5#-GCCCATAGCAAAAGCATGAAAG-3# 54 C 30 sec, 72 C 45 sec) 35 cycles–(72 C 5 min) (ch22:22,361,683-22,361,704) reverse primer: 5#-ATACAGTCGTGATGGGAAAGGG-3# (ch22:22,362,768-22,362,789) forward primer: (94 C 5 min)–(94 C 30 sec, SWI/SNF related, 5#-GGTGTCCTGTGTCCTCCAGC-3# matrix associated, 55 C 30 sec, 72 C 30 sec) actin dependent (ch22:22,469,412-22,469,431) 40 cycles–(72 C 5 min) regulator of reverse primer: chromatin, 5#-GAAACGGGACTGTTCCCACG-3# subfamily b, (ch22:22,470,214-22,470,233) member 1 D-dopachrome forward primer: (94 C 5 min)–(94 C 30 sec, 61 C 30 sec, 72 C 30 sec) tautomerase 5#-TCTAATAGGGCTAGGAACCG-3# 35 cycles–(72 C 5 min) (ch22:22,641,496-22,641,515) reverse primer: 5#-AGACCCTGATGTGTCCTTAC-3# (ch22: 22,640,396-22,640,415) Adenosine forward primer: (94 C 5 min)–(94 C 30 sec, A2a receptor 5#-ACCTGCAGAACGTCACCAACT 54 C 30 sec, 72 C 45 sec) ACT-3# (ch22:23,154,033-23,154,056) 35 cycles–(72 C 5 min) reverse primer: 5#-CCATGCCGAGTAATT CAGCCTCT-3# (ch22:23,154,984-23,155,006) Ral-GDS related protein Rgr
Size of PCR product (bp)b 1107
822
1120
974
Abbreviations: min, minutes; sec, seconds. The sequence information described in this table was collected in November, 2004 using the Ensembl Genome Database (http://www.ensembl.org/). a Numbers within parentheses denote the coordinates of each primer on chromosome 22. b Expected size of each PCR product calculated according to Human Genome Sequence Build 35.
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described previously [31]. We selected the probe sets whose differences in signal intensity were less than 2.0-fold in this comparison as probe sets with reproducibility of signal intensity, as described elsewhere [32]. Using the probe sets with reproducibility of signal intensity, we next performed two pairwise comparisons between the data sets of the NK sample (n 5 2) and the MRTK tumor sample (n 5 1). In the present study, to get a picture of the gene expression profile of MRTK, we consequently selected probe sets showing a marked alteration of signal intensity that was more than 4.0-fold higher or lower in the MRTK tumor sample than in the NK sample in all pairwise comparisons, as described previously [31]. All the procedures used for selection of probe sets were performed using GeneSpring 4.2 software (Agilent Technologies, Palo Alto, CA). 2.7. Functional classification of genes Functional classification of genes was performed according to the classification of the Gene Ontology Consortium (http://www.geneontology.org) and iterative PubMed search (National Center for Biotechnology Information– NCBI, Bethesda, MD) [33].
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3. Results 3.1. Molecular genetic alterations of chromosomal region 22q11.23 in MRTK cells To investigate the molecular genetic alterations in MRTK in our patient, we first analyzed the molecular cytogenetic alterations in chromosomal region 22q11.23 in MRTK cells using FISH analysis. Interphase FISH analysis using a BAC probe RP11-698L6 or RP11-80O7 showed the absence of signals for these two probes in O995 out of 1,000 tumor cell nuclei examined, and two signals for RP11-423E19 at 22q13.33 (green signals) were present in all 1,000 tumor cell nuclei examined (Fig. 1). Interphase FISH analysis using a BAC probe RP11-61N10 or RP11297B9 revealed the presence of two signals (orange signals) for each probe in O960 out of 1,000 tumor cell nuclei examined (Fig. 1). To confirm the results obtained by FISH analyses, we investigated the genomic region at 22q11.23 in MRTK cells using Southern blot analysis. As shown in Fig. 2, Southern blot analyses using Rgr, SMARCB1, and DDT probes detected a homozygous deletion of these genes in MRTK cells. Southern blot analyses for BCR and ADORA2A
Fig. 1. Schematic representation of chromosomal deletion region at 22q11.23 in malignant rhabdoid tumor of the kidney (MRTK) cells in our patient. The FISH signal patterns obtained using BAC probes for chromosomal region 22q11.23 (orange signals indicated by arrows) and the RP11-423E19 probe (green signals indicated by arrowheads) are shown with a map of chromosomal region 22q11.23. The position of each BAC within chromosomal region 22q11.23 is denoted as a black bar with an approximate sequence position on chromosome 22 according to the information in Ensembl Genome Database (http://www.ensembl.org/). The broken line indicates an ~0.29-Mb homozygously deleted region found in MRTK cells, that was confirmed by FISH and Southern blot analyses (see Fig. 2). Coordinates of genes and BACs on chromosome 22 are shown in megabases from 22ptel. Abbreviations: MRTK, malignant rhabdoid tumor of the kidney. a Coordinates of known genes located within or around the homozygously deleted region at 22q11.23 found in MRTK cells in our patient. b Positions of BACs in chromosomal region 22q11.23.
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Fig. 2. Southern blot analyses of malignant rhabdoid tumor of the kidney. Numbers on the left side of M show the size marker. (A) Rgr gene. (B) SMARCB1 gene. (C) DDT gene. (D) BCR gene. (E) ADORA2A gene. Capital letters at the top of each lane or after GL refer to the restriction enzymes used: B, BamHI; E, EcoRI; GII, BglII; K, KpnI; S, SacI. Other abbreviations: GL, germline band; M, size marker; MRTK, DNA derived from malignant rhabdoid tumor of the kidney; NC, normal control DNA derived from peripheral blood mononuclear cells of healthy volunteers.
detected neither rearranged bands nor diminished signals for germline bands (Fig. 2). Considering the results obtained by FISH analyses and the sequence information of this genomic region, the results presented here indicate homozygous deletion of an ~0.29-Mb genomic region bordered by Rgr and DDT and the retention of genomic regions detected with BCR and ADORA2A probes for Southern blot analysis, in MRTK cells in our patient (Figs. 1 and 2).
3.2. Genes differentially expressed between NK sample and MRTK tumor sample To investigate the transcriptional profile of MRTK, we analyzed the differences in gene expression profile between the NK sample and MRTK tumor sample. We first selected 1,020 probe sets as those with reproducibility of signal intensity in this experiment, as described in Section 2.6. Among 1,020 probe sets selected, we obtained 25 probe sets corresponding to 25 genes showing more than 4.0-fold higher expression in the MRTK tumor sample than in the NK sample and 64 probe sets corresponding to 64 genes showing more than 4.0-fold lower expression in the MRTK tumor sample than in the NK sample (Table 2). Among the 64 genes showing decreased expression in the MRTK tumor sample, MIF and DDT genes, which are mapped on the homozygously deleted region found in our case and are included in the G110 Cancer Array used in our
experiment, were found to display lower expression in the MRTK tumor sample (Table 2; Figs. 1 and 2). The detailed functional classifications of genes showing increased or decreased expression in the MRTK tumor sample are also summarized in Table 2.
4. Discussion MRTK is a relatively uncommon solid tumor in children, compared with other types of pediatric cancer, and knowledge about the molecular signature of this tumor is limited. We therefore analyzed the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in our patient. Using FISH and Southern blot analyses, we found that an ~0.29-Mb genomic region bordered by the Rgr and DDT genes was homozygously deleted in these MRTK cells (Figs. 1 and 2). Recent reports regarding the aberrant genomic organization of chromosomal region 22q11.23 in MRT cells suggested the existence of a homozygous deletion within the genomic region bordered by sequence-tagged sites (STSs) D22S556 and D22S42 in some cases of MRT, and the SMARCB1 gene, a candidate tumor suppressor gene, located within this region has been found to show frequent, biallelic inactivating alterations in MRT cells [14,15,17,19–22]. As shown in Figs. 1 and 2, this gene was also inactivated by a homozygous deletion in the MRTK in our patient, and this finding supports the notions
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Table 2 Functional classification of 89 genes showing more than 4.0-fold higher (25 genes) or lower (64 genes) expression in MRTK tumor sample than in noncancerous kidney (NK) sample
Biological function
Genes showing increased expression in MRTK (MRTKONK; 25 genes)
Signal transduction
WNT5A, DDR2, MDK, TGFB3, FZD2
Cell cycle
DTYMK, MKI67, ABL1, PCNA, MAD2L1, PRKDC, CCNE1, TK1, TP53, GAS1 IGF2, IGFBP6, EMP1, TFRC, ABL1 IGF2, MKI67, MDK, NAP1L1, NME1
Cell growth Cell proliferation Cell metabolism Cell adhesion
DDR2
Transcription Apoptosis Immune response DNA replication DNA repair Stress response Cell-cell communication DNA metabolism RNA metabolism DNA damage response Unfolded protein binding activity Protein transport Protein phosphatase regulator activity Transporter activity Muscle contraction Unknown fuction
CRABP2, NFIB
PCNA, TOP2A, NAP1L1 PRKDC TGFB3 TK1 HNRPU ABL1
Genes showing decreased expression in MRTK (NKOMRTK; 64 genes) IL1R1, EGFR, PTHR1, MUC1, NR4A1, NR4A2, PLCL1, TACSTD1, FGFR4, GDF15, PLCG2, EDNRA, S100A2, FGF9, FKBP1B, ERBB3, DAB2, FGF1, BCR, PDGFRB, TACSTD2, PTPRK DUSP1, GADD45A, FOS, ACPP PTN, EGFR, PTHR1, IGFBP7, IGFBP2, PDGFRB EDNRA, FGF9, EGFR, ERBB3, DAB2, FGF1 HYAL1, MMP7, DDT, MT1G, GPX3, ENPP1, PLCG2, PCCA, ACP5, GSTM3, ASAH1 TACSTD1, CDH1, ITGA1, CCL2, LAMB1, CX3CL1, ICAM2, PTPRK, NID2 NR4A1, ESRRA, FOS, NR4A2, CEBPD, HOXD4 BAD, TNFSF10, GADD45A, CASP4 MIF, CD24, MX2, IFITM2 GADD45A DUSP1, SGK PTHR1
HSPA2 CAV2 PPP2R3A ABCB1 MYH11 TM4SF1, C5orf18
Abbreviations: NK, noncancerous kidney; MRTK, malignant rhabdoid tumor of the kidney. Genes are described using gene symbols.
suggested by other authors. In addition, according to information on the human genome sequence, several genes and putative transcriptional units other than SMARCB1 have recently been identified within the homozygously deleted region found in our case (Ensembl Genome Database, http:// www.ensembl.org/). Further characterization of this genomic region in more MRTK tumors may provide additional information on genes affecting the biological characteristics of this tumor. Using a high-density oligonucleotide DNA array, we found 25 genes showing more than 4.0-fold higher expression in the MRTK tumor sample and 64 genes showing more than 4.0-fold lower expression in the MRTK tumor sample, than in the NK sample (Table 2). The decreased expression of MIF and DDT found in our case was suspected to be the result of homozygous deletion of a genomic region at 22q11.23 in the MRTK cells during tumorigenesis, although we failed to analyze the status of this chromosomal region in the patient’s noncancerous cells because samples for FISH and DNA analysis were not available (Table 2; Figs. 1 and 2). The 25 genes showing increased expression in the MRTK tumor sample included genes regulating the cell cycle (10 genes: DTYMK, MKI67, ABL1, PCNA, MAD2L1,
PRKDC, CCNE1, TK1, TP53, GAS1), DNA replication (3 genes: PCNA, TOP2A, NAP1L1), and cell proliferation (5 genes: IGF2, MKI67, MDK, NAP1L1, NME1) (Table 2). Among these genes, increased expression of p53 protein in MRTK cells has been reported to be associated with aggressive biological behavior of this tumor [34]. Increased expression of MKI67, PCNA, and NAP1L1 has been proved to be related to progression of cell growth [35–37], suggesting acceleration of cell division and tumor cell growth in this MRTK tumor. The increased expression of genes regulating cell growth (5 genes: IGF2, IGFBP6, EMP1, TFRC, ABL1) in this MRTK tumor sample also suggests alterations of signaling for cell growth in this MRTK tumor [38–41] (Table 2). Thus, the results obtained in the present study suggest that an altered pattern of signaling for tumor cell growth may have contributed to the biological characteristics of the MRTK tumor in our patient and may have led to the activation of tumor cell growth. The 64 genes showing decreased expression in the MRTK tumor sample included genes regulating apoptosis (4 genes: BAD, TNFSF10, GADD45A, CASP4) (Table 2). Among these changes, decreased expression of TNFSF10 in MRTK tumors has been reported previously [42]. Alterations of expression of genes regulating apoptosis in MRTK
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tumors have been reported by other authors, and some of them have been proposed as therapeutic targets for MRTK tumors [42,43]. Thus, our results support the previously described notion and may provide additional information on the expression profile of genes regulating apoptosis in MRTK tumors [42,43]. In the present study, we found that IGF2, IGFBP6, and MDK showed increased expression, whereas IGFBP2, IGFBP7, and PTN showed decreased expression in the MRTK tumor sample (Table 2). Among these genes, IGF2, IGFBP2, IGFBP6, and IGFBP7 are members of the IGF-axis, and MDK and PTN are members of the midkine family of growth and differentiation factors [38,44]. Alteration of the expression of IGF2 and MDK has been shown in some MRTK tumors and/or cell lines, and these changes are considered to affect the biological characteristics of this tumor [10,45–49]. A recent report has also indicated direct control of PTN expression by SMARCB1 protein via transcriptional activation in an MRT cell line [50]. Thus, the altered pattern of expression of some IGFaxis members and members of the midkine family of growth and differentiation factors found in the present study supports the significance of these alterations in the biological characteristics of MRTK. A recent report describing gene expression profiling of pediatric CNS embryonal tumors, including MRTs, has shown that some genes expressed during myogenesis, such as TPM2, CNN2, NFATC4, and MRCL3, were included in the set of genes discriminating MRTs from other pediatric CNS tumors, and the authors therefore suggested a mesenchymal origin for MRTs [51]. We could not find differential expression of such marker genes between the NK sample and MRTK tumor sample, because the probe sets for marker genes described in the previous report were not included in the microarray used in our study (Table 2). Further analyses of the expression profile of more genes in MRTK tumors may provide a clue to understand the histogenetic origin of this tumor. In summary, we analyzed the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in our patient. FISH and Southern blot analyses of chromosomal region 22q11.23 revealed a homozygous deletion of an ~0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with the NK sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case
may have contributed to the biological characteristics of the MRTK tumor that arose in our patient.
Acknowledgments This work was supported in part by a Grant from the Ministry of Education, Culture, Sports, Science, and Technology to promote advanced scientific research to Nihon University, School of Medicine, by Grant-in-Aids from the Ministry of Education, Science, Sports and Culture of Japan (no. 17591119 and 16591359), by a Nihon University Research Grant for Assistants and Young Researchers (no. 05-031), and by a Nihon University Joint Research Grant (no. 05-016).
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