Identification of SII as a New Fusion Partner Gene Tumors with and without Chromosome 8q12 Abnormalities : Activation in Salivary Gland

We have previously shown (K. Kas et al., Nat. Genet., 15: 170 –174, 1997) that the developmentally regulated zinc finger gene pleomorphic adenoma gene 1 (PLAG1) is the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between PLAG1 and the constitutively expressed gene for b-catenin (CTNNB1), leading to activation of PLAG1 expression and reduced expression of CTNNB1. Here we have studied the expression of PLAG1 by Northern blot analysis in 47 primary benign and malignant human tumors with or without cytogenetic abnormalities of 8q12. Overexpression of PLAG1 was found in 23 tumors (49%). Thirteen of 17 pleomorphic adenomas with a normal karyotype and 5 of 10 with 12q13–15 abnormalities overexpressed PLAG1, which demonstrates that PLAG1 activation is a frequent event in adenomas irrespective of karyotype. In contrast, PLAG1 was overexpressed in only 2 of 11 malignant salivary gland tumors analyzed, which suggests that, at least in salivary gland tumors, PLAG1 activation preferentially occurs in benign tumors. PLAG1 overexpression was also found in three of nine mesenchymal tumors, i.e., in two uterine leiomyomas and one leiomyosarcoma. RNase protection, rapid amplification of 5*-cDNA ends (5*-RACE), and reverse transcription-PCR analyses of five adenomas with a normal karyotype revealed fusion transcripts in three tumors. Nucleotide sequence analysis of these showed that they contained fusions between PLAG1 and CTNNB1 (one case) or PLAG1 and a novel fusion partner gene, i.e., the gene encoding the transcription elongation factor SII (two cases). The fusions occurred in the 5* noncoding region of PLAG1, leading to exchange of regulatory control elements and, as a consequence, activation of PLAG1 gene expression. Because all of the cases had grossly normal karyotypes, the rearrangements must result from cryptic rearrangements. The results suggest that in addition to chromosomal translocations and cryptic rearrangements, PLAG1 may also be activated by mutations or indirect mechanisms. Our findings establish a conserved mechanism of PLAG1 activation in salivary gland tumors with and without 8q12 aberrations, which indicates that such activation is a frequent event in these tumors. INTRODUCTION We have previously identified (1, 2) a new, developmentally regulated zinc finger gene, designated PLAG1, as the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The translocation results in promoter swapping between PLAG1 and the constitutively expressed gene for b-catenin (CTNNB1) in 3p21, leading to activation of PLAG1 expression and reduced expression of CTNNB1. The breakpoints invariably occur in the 59 noncoding regions of both genes. The deduced PLAG1 protein contains seven NH2-terminal C2H2 zinc finger domains and a serinerich COOH terminus, which acts as a transcriptional activator (3). PLAG1 is developmentally regulated with expression mainly restricted to certain fetal tissues. b-catenin is a protein functioning as an interface in adherens junctions and in the WG/WNT signaling pathway (4, 5). b-catenin has also been implicated in tumorigenesis (4). Recently (6), we identified a second translocation partner gene of PLAG1 in pleomorphic adenomas with a recurrent t(5;8)(p13;q12) translocation, namely LIFR. LIFR encodes the ubiquitously expressed receptor for the leukemia inhibitory factor (7). The translocation results in up-regulation of PLAG1 gene expression under control of the LIFR promoter, i.e., a mechanism similar to that seen in adenomas with 3;8-translocations. In addition to the above-mentioned subgroup of pleomorphic adenomas with abnormalities involving 8q12 (39% of the cases), there are at least three other cytogenetic subgroups that are characterized by (i) rearrangements of 12q13–15 (8% of the cases); (ii) sporadic clonal changes not involving 3p21, 8q12 or 12q13–15 (23% of the cases); and (iii) an apparently normal karyotype (30% of the cases; Refs. 8–10). The gene consistently rearranged in adenomas with 12q13–15 abnormalities is the high mobility group protein gene, HMGIC (11– 13). This gene is also rearranged in a variety of benign mesenchymal tumors with 12q13–15 abnormalities (11, 14–16). Our previous studies of PLAG1 were restricted to pleomorphic adenomas with 8q12 abnormalities (1, 6). Here we present results showing that PLAG1 activation is not confined to adenomas with 8q12 abnormalities but is also found in tumors with normal karyotype and 12q13–15 abnormalities as well as in individual cases of malignant salivary gland tumors and mesenchymal tumors. In addition, we show that PLAG1 may also be activated by cryptic rearrangements in cases with normal karyotypes, leading to fusions between PLAG1 and CTNNB1 or a novel fusion partner gene. MATERIALS AND METHODS Tumor Material and Cytogenetic Analysis. Fresh tumor tissue was obtained from patients at the time of surgery. Chromosome preparations were made from short-term primary cultures as described previously (17). FortyReceived 9/11/98; accepted 12/14/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Swedish Cancer Society; the IngaBritt and Arne Lundbergs Research Foundation; the Assar Gabrielssons Research Foundation; the EC through Biomed 1 program “Molecular Cytogenetics of Solid Tumours”; the “Geconcerteerde Onderzoekacties 1997–2001”; the “Fonds voor Wetenschappelijk Onderzoek Vlaanderen”; and the “ASLK-programma voor Kankeronderzoek.” K. K. is a postdoctoral scholar of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen. 2 Both authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Laboratory of Cancer Genetics, Department of Pathology, Göteborg University, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. Phone: 46-31-3422922; Fax: 46-31-820525; E-mail: [email protected]. 4 The abbreviations used are: PLAG1, pleomorphic adenoma gene 1; UTR, untranslated region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene; RT-PCR, reverse transcription-PCR; 59-RACE, rapid amplification of 59-cDNA ends.