Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

PLAG1, the main translocation target in pleomorphic adenoma of the salivary glands, is a positive regulator of IGF-II.

PLAG1, a novel developmentally regulated C2H2 zinc finger gene, is consistently rearranged and overexpressed in pleomorphic adenomas of the salivary glands with 8q12 translocations. In this report, we show that PLAG1 is a nuclear protein that binds DNA in a specific manner. The consensus PLAG1 binding site is a bipartite element containing a core sequence, GRGGC, and a G-cluster, RGGK, separated by seven random nucleotides. DNA binding is mediated mainly via three of the seven zinc fingers, with fingers 6 and 7 interacting with the core and finger 3 with the G-cluster. In transient transactivation assays, PLAG1 specifically activates transcription from its consensus DNA binding site, indicating that PLAG1 is a genuine transcription factor. Potential PLAG1 binding sites were found in the promoter 3 of the human insulin-like growth factor II (IGF-II) gene. We show that PLAG1 binds IGF-II promoter 3 and stimulates its activity. Moreover, IGF-II transcripts derived from the P3 promoter are highly expressed in salivary gland adenomas overexpressing PLAG1. In contrast, they are not detectable in adenomas without abnormal PLAG1 expression nor in normal salivary gland tissue. This indicates a perfect correlation between PLAG1 and IGF-II expression. All of these results strongly suggest that IGF-II is one of the PLAG1 target genes, providing us with the first clue for understanding the role of PLAG1 in salivary gland tumor development.

Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.

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 beta-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 over-expression 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.

fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as an antisense sequence in the Finkel-Biskis-Reilly murine sarcoma virus.

The Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) is capable of inducing osteosarcomas in susceptible mice. This retrovirus transduced sequences derived from the transcription factor c-fos and from an unrelated mouse sequence called fox. Here, we describe the cloning and sequence analysis of human and mouse cellular cDNAs hybridizing to the fox sequence. The cloned cDNAs encode for a single ubiquitin-like (Fubi) protein fused in frame to S30, a protein of the small ribosomal subunit. Fubi conserved amino acid residues known to be involved in the ATP-dependent proteolytic activity of ubiquitin. Moreover, the fau gene is conserved in several species, while its mRNA is ubiquitously expressed in different mouse tissues. Surprisingly, FBR-MuSV transduced the complete but mutated open reading frame (ORF) in its reversed transcriptional orientation. This is the first report about a retrovirus in which an antisense sequence to a cellular gene, which we called fau (FBR-MuSV-associated ubiquitously expressed gene), is discovered. Rat-2 cells transfected with plasmids containing v-fau/fox recombinants of FBR-MuSV revealed a twofold increase of the transformation capacity of FBR-MuSV 'in vitro' because of the fau antisense sequence. Newly formed retropseudogenes were identified in three out of eight primary radiation-induced osteosarcomas. This high incidence of creating retropseudogenes in these 90Sr-induced bone tumours may contribute to the mechanism by which FBR-MuSV, originally isolated from such tumours, acquired the fau gene in its reverse orientation.

The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter.

We have previously shown that the PLAG1 gene on chromosome 8q12 is consistently rearranged in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between the PLAG1 gene, which encodes a novel zinc finger protein, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein with roles in cell-cell adhesion and the WG/WNT signalling pathway. In order to assess the importance of other translocation partner genes of PLAG1, and their possible relationship to CTNNB1, we have characterized a second recurrent translocation, i.e. the t(5;8)(p13;q12). This translocation leads to ectopic expression of a chimeric transcript consisting of sequences from the ubiquitously expressed gene for the leukemia inhibitory factor receptor (LIFR) and PLAG1. As for the t(3;8), the fusions occurred in the 5'-noncoding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. The results of the current as well as previous studies indicate that ectopic expression of PLAG1 under the control of promoters of distinct translocation partner genes is a general pathogenetic mechanism for pleomorphic adenomas with 8q12 aberrations.

Characterization of the mouse Men1 gene and its expression during development.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

Characterization of a processed pseudogene of human FAU1 on chromosome 18.

A member of the human FAU (Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed) gene subfamily, encoding the ribosomal protein S30 fused in frame to an ubiquitin-like protein, was cloned, sequenced and analysed. This clone, FAU1P, is a processed pseudogene with a completely intact, although transcriptionally silent, open reading frame of 137 codons. FAU1P exhibits an amplification of the (AAG) triplet repeat present in the S30 coding part of FAU. FAU1P is integrated in an antisense orientation within a sequence homologous to the promoter of the islet amyloid polypeptide (IAPP or amylin)-encoding gene. By means of PCR hybrid panel mapping, FAU1P was assigned to chromosome 18.

On the technicalities of discovering and applying protein biomarkers for cancer prevention.

Primary prevention of cancer is one of the key approaches used to combat cancer. By identifying people at high risk of developing cancer, it becomes possible to develop intervention efforts on prevention rather than treatment. Prevention includes avoiding exposure to known cancer-causing agents, enhancement of host-defence mechanisms, modifying lifestyle and chemoprevention. The latter is the use of specific agents to suppress or reverse carcinogenesis and thereby prevent the development of cancers. Understanding primary molecular events in tumour development is therefore the key. The causes of cancer first produce reversible changes in the target cells, and a long interval is expected between cancer induction and the presentation of disease. Up to now, we had no reliable biomarkers to monitor these primary events, to select high-risk groups and individuals likely to get cancer, and to monitor preventive treatments or strategies to normalize these markers and prevent the individual from getting cancer. Recent developments in proteomic research, however, promise to deliver on these major needs. We here describe the technological armatorium of, and the recent advances in the field of protein biomarker discovery and discuss the future use of protein biomarkers for (1) reliable identification of high-risk groups; (2) clinical guidance of preventive strategies; and (3) elucidation of the mechanism of action for novel (natural product) prevention therapies and regimens.

[Molecular techniques lead to the first insights into the pathophysiology of salivary gland adenomas]. / Moleculair armatorium leidt tot de eerste inzichten in de pathofysiologie van speekselklieradenomen.

Pleomorphic adenomas are the most common type of salivary gland tumours. Activation of the PLAG1 gene on chromosome 8q12 is the most frequent mutation found in these tumours. This results from chromosomal translocations leading to promoter substitution between PLAG1, mainly expressed in fetal tissue, and more broadly expressed genes. The replacement of the PLAG1 promoter, inactive in adult salivary glands, by a strong promoter derived from the translocation partner, leads to ectopic expression of PLAG1 in the tumor cells. This abnormal PLAG1 expression results in deregulation of PLAG1 target genes causing salivary gland tumorigenesis. PLAG1 binds to promoter 3 of the Insulin-like growth factor 2 gene (IGF2) and stimulates its activity. IGF2 is highly expressed in salivary gland adenomas overexpressing PLAG1 while no IGF2 expression is found in adenoma without abnormal PLAG1 expression nor in normal salivary gland tissue, indicating a perfect correlation between PLAG1 and IGF2 expression. These results provide us with the first clue for understanding the role of PLAG1 in salivary gland tumor development. IGF2 perfectly fits in the picture of a restarted developmental program with concomitant loss of differentiation, the typical hallmark for any tumour. Salivary gland genesis provides a system for studying the development of glandular organs having many basic features in common with the salivary gland, such as breast, kidney, lung, pancreas and prostate. With a unique salivary gland organ culture system we now can study principles of epitheliogenesis, tubulogenesis and branching morphogenesis. Genes expressed at the spot where during tumourigenesis proliferation overrules differentiation constitute new targets for reverting the proliferative, tumour-specific stage. By elucidating molecular mechanisms involved in human cancer, we will hence contribute at the level of fundamental cancer research (oncogenesis) and normal organ development (organogenesis).

Genomic structure and expression of the human fau gene: encoding the ribosomal protein S30 fused to a ubiquitin-like protein.

The fau gene is the cellular homolog of the fox sequence in the Finkel-Biskis-Reilly Murine Sarcoma Virus (FBR-MuSV). This virus acquired the fau sequence in its reversed transcriptional orientation. Human and mouse fau cDNA's were identified and both encode a new protein of 133 AA. We show that fau (for FBR-MuSV associated ubiquitiously expressed gene) becomes expressed in all different tissues tested as a 600 bp messenger and we report the genomic structure of the human fau gene. The gene consists of five exons and four introns and the 5' untranslated region displays characteristic features for a housekeeping gene. Fau encodes the ribosomal protein S30 fused to a Ubiquitin-like protein.

First insights into the molecular basis of pleomorphic adenomas of the salivary glands.

Pleomorphic adenoma, or mixed tumor of the salivary glands, is a benign tumor originating from the major and minor salivary glands. Eighty-five percent of these tumors are found in the parotid gland, 10% in the minor (sublingual) salivary glands, and 5% in the submandibular gland. It is the most common type of salivary gland tumor, accounting for almost 50% of all neoplasms in these organs. In fact, after the first observation of recurrent loss of chromosome 22 in meningioma, this was the second type of benign tumor for which non-random chromosomal changes were reported. The rate of malignant change with the potential to metastasize has been reported to be only 2 to 3%, and only a few cases of metastasizing pleomorphic salivary gland adenomas have been described to date. The fact that these tumors arise in organs located in an ontogenetic transitional zone, a region where endoderm and ectoderm meet, might be one of the reasons for the often-problematic histopathological classification. This type of benign tumor has been cytogenetically very well-characterized, with several hundreds of tumors karyotyped. In addition to the cytogenetic subgroup with an apparently normal diploid stemline (making up approximately 30% of the cases), three major cytogenetic subgroups can be distinguished. In addition to a subgroup showing non-recurrent clonal abnormalities, another subgroup is various translocations involving 12q15. By far the largest cytogenetic subgroup, however, consists of tumors with chromosome 8 abnormalities, mainly showing translocations involving region 8q12. The most frequently encountered aberration in this group is a t(3;8)(p21;q12).

Isolation, cDNA, and genomic structure of a conserved gene (NOF) at chromosome 11q13 next to FAU and oriented in the opposite transcriptional orientation.

In our effort to characterize a gene at chromosome 11q13 involved in a t(11;17)(q13;q21) translocation in B-non-Hodgkin lymphoma, we have identified a novel human gene, NOF (Neighbour of FAU). It maps right next to FAU in a head to head configuration separated by a maximum of 146 nucleotides. cDNA clones representing NOF hybridized to a 2. 2-kb mRNA present in all tissues tested. The largest open reading frame appeared to contain 166 amino acids and is proline rich, and the sequence shows no homology with any known gene in the public databases. The NOF gene consists of 4 exons and 3 introns spanning approximately 5 kb, and the boundaries between exons and introns follow the GT/AG rule. The NOF locus is conserved during evolution, with the predicted protein having over 80% identity to three translated mouse and rat ESTs of unknown function. Moreover, the mouse ESTs map in the same organization, closely linked to the FAU gene, in the mouse genome. NOF, however, is not affected by the t(11;17)(q13;q21) chromosomal translocation.

Mapping of the 8q12 translocation breakpoint to a 40-kb region in a pleomorphic adenoma with an ins(8;3)(q12;p21.3p14.1).

The translocation t(3;8)(p21;q12) is the most common chromosome abnormality observed in pleomorphic adenomas of the salivary glands. In this paper we describe the physical mapping of the breakpoints in an adenoma with a variant t(3;8), viz., an ins(8;3)(q12;p21.3p14.1). Using sequence-tagged sites (STSs) corresponding to landmarks within a previously identified yeast artificial chromosome (YAC) spanning the breakpoint in adenomas with t(3;8), cosmids isolated from a chromosome 8-specific cosmid library. The 8q12 insertion breakpoint was mapped by FISH to a 300-kb region flanked by MOS and a new STS, CH129. A cosmid within this region was shown to span the breakpoint. To test whether the recently identified FHIT gene, which maps to 3p14.2, was disrupted by the 3p rearrangement, we also isolated an FHIT YAC and mapped this YAC by FISH distal to the most proximal 3p breakpoint. In addition, RT-PCR analysis revealed only a normal-sized FHIT transcript, suggesting that FHIT is not affected by the 3;8-rearrangement.

Identification of a yeast artificial chromosome spanning the 8q12 translocation breakpoint in pleomorphic adenomas with t(3;8)(p21;q12).

A subgroup of pleomorphic adenomas of the salivary glands is characterized by translocations involving chromosome 8, with consistent breakpoints at 8q12. As part of a positional cloning effort to isolate the gene(s) affected by these translocations we now report the mapping of the 8q12 breakpoint in two primary pleomorphic adenomas with the recurrent t(3;8)(p21;q12). Yeast artificial chromosome (YAC) clones corresponding to eight different loci in 8q11-12 were isolated and mapped by fluorescence in situ hybridization (FISH). The t(3;8) breakpoint was mapped within a 1 Mb region flanked by MOS proximally and by the genetic marker D8S166 distally. One YAC within this region was shown to span the t(3;8) breakpoint in two tumors. This YAC will provide an excellent tool for isolating the gene(s) at the breakpoint(s) in adenomas with t(3;8).

Transcriptional activation capacity of the novel PLAG family of zinc finger proteins.

We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.

Identification and molecular characterization of TM7SF2 in the FAUNA gene cluster on human chromosome 11q13.

In this report, the identification and molecular characterization of a novel gene, designated TM7SF2, is reported. This gene was found in the FAU neighboring area (FAUNA) to which other genes have been mapped previously. The FAUNA gene cluster is located at chromosome 11q13 between landmarks H4B and D11S2196E. The TM7SF2 gene contains eight coding exons, and their splice site consensus sequences are consistent with AG/GT rule. Northern blot analysis with a cDNA probe corresponding to TM7SF2 revealed varying expression levels of a 1.7-kb transcript in adult human heart, brain, pancreas, lung, liver, skeletal muscle, kidney, ovary, prostate, and testis, but no detectable expression in placenta, spleen, thymus, small intestine, colon (mucosal lining), or peripheral blood leukocytes. The open reading frame in the cDNA sequence codes for a protein of 590 amino acids that is rich in glycine (23%) and arginine (17%) residues in its amino-terminal half and contains seven transmembrane domains in its carboxy-terminal half. The transmembrane region of the putative TM7SF2 protein shows amino acid sequence similarity to those of the lamin B receptor and the C14/C24 sterol reductase.

Fluorescence in situ hybridization mapping of breakpoints in pleomorphic adenomas with 8q12-13 abnormalities identifies a subgroup of tumors without PLAG1 involvement.

Recently, we identified the PLAG1 gene as the target gene in pleomorphic adenomas with chromosome abnormalities involving 8q12. The majority of breakpoints were shown to reside within the 5' noncoding region of the gene. We now report three pleomorphic adenomas with breakpoints located distal to PLAG1 in band 8q13. These tumors had the following chromosome 8 abnormalities: ins(8;12)(q12-13;q14q15), t(8;12)(q13;q15), and t(6;8)(p21.3-22;q13). Fluorescence in situ hybridization mapping of the chromosome 8 breakpoints revealed a yeast artificial chromosome clone spanning the breakpoints in two tumors. In none of the cases was PLAG1 activated and/or disrupted. Three candidate genes, N8, HMGIC, and HMGIY, were analyzed for rearrangements and/or abnormal expression by using reverse transcriptase-polymerase chain reaction, rapid amplification of 3' cDNA ends, and Northern blot analyses.

Assignment of the human proprotein convertase gene PCSK5 to chromosome 9q21.3.

The human PCSK5 gene, which encodes a subtilisin-like proprotein processing enzyme, has been mapped by analysis of somatic cell hybrids and YAC clones as well as fluorescence in situ hybridization to chromosome 9q21.3 near markers D9S175 and D9S276.

A 2-Mb YAC contig and physical map covering the chromosome 8q12 breakpoint cluster region in pleomorphic adenomas of the salivary glands.

Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands. Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12. In previous studies, we have shown that these breakpoints are located in a 9-cM interval between MOS/D8S285 and D8S260. Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285. Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively. Initial fluorescence in situ hybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12. This contig has at least double coverage and consists of 34 overlapping YAC clones. The localization of the YACs was confirmed by FISH analysis. On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig. The relative positions of various known genes and expressed sequence tags within this contig were also determined. Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between the MOS proto-oncogene and STS EM156. The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be the PLAG1 gene, which encodes a novel zinc finger protein.