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.

Histologic localization of PLAG1 (pleomorphic adenoma gene 1) in pleomorphic adenoma of the salivary gland: cytogenetic evidence of common origin of phenotypically diverse cells.

Pleomorphic adenoma gene 1 (PLAG1), a zinc finger transcription factor gene, is consistently rearranged and overexpressed in human pleomorphic adenomas of the salivary glands with 8q12 translocations. In this report, we describe the immunohistochemical localization of PLAG1 protein in pleomorphic adenomas of the salivary gland and corresponding normal tissue, in relation to cytokeratin, vimentin, and BCL-2 expression. Normal salivary gland tissue was not immunoreactive for PLAG1. In primary pleomorphic adenomas, cells strongly immunoreactive for PLAG1 were detected in the outer layer of tubulo-ductal structures, which are thought to be the origin of cells with bi-directional, epithelial, and mesenchymal phenotypes. In contrast, epithelial cells with abundant cytokeratin in the inner tubulo-ductal structures only sporadically expressed PLAG1. BCL-2 immunoreactivity was found mainly in the cells surrounding the tubulo-ductal structures and in the solid undifferentiated cellular masses, within the areas that had moderate PLAG1 immunoreactivity. The variability of PLAG1 expression in neoplastic cells seemed to reflect the morphologic heterogeneity that correlated with the stage of differentiation of the tumor cells. Immunohistochemical/cytogenetic evaluation of two pleomorphic adenomas with t(3;8)(p21;q12) or t(5;8)(p13;q12) translocations demonstrated the clonal nature of immunophenotypically diverse cells. This finding confirms the theory that pleomorphic adenoma cells share a common single-cell origin, most likely from the epithelial progenitor basal duct cells.

Menin interacts directly with the homeobox-containing protein Pem.

The tumour suppressor gene causing multiple endocrine neoplasia type 1 (MEN1) encodes a 610 amino acid protein, menin. In order to identify menin-interacting proteins we used a yeast two-hybrid assay to screen a 12.5-dpc mouse embryo library with partial menin encompassing amino acids 278 to 476. This identified a homeobox containing protein encoded by a placenta and embryonic expression gene, referred to as Pem. GST-pull-down and coimmunoprecipitation experiments confirmed the interaction. Both proteins colocalised predominantly in the nucleus but were occasionally also found in the cytoplasm. Furthermore, in situ hybridisation studies revealed similarities in their expression patterns in mouse embryos and adult tissues. In adult mice both Men1 and Pem yielded strong signals in testis, Sertoli cells and particularly in seminiferous tubules. Thus, our study has identified that menin interacts with Pem, and the high expression of these proteins in the testis suggests a role in spermatogenesis.

[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).

ESE-3, a novel member of an epithelium-specific ets transcription factor subfamily, demonstrates different target gene specificity from ESE-1.

Most cancers originate as a result of aberrant gene expression in mainly glandular epithelial tissues leading to defects in epithelial cell differentiation. The latter is governed by distinct sets of transcriptional regulators. Here we report the characterization of epithelium-specific Ets factor, family member 3 (ESE-3), a novel member of the ESE subfamily of Ets transcription factors. ESE-3 shows highest homology to two other epithelium restricted Ets factors, ESE-1 and ESE-2. ESE-3, like ESE-1 and ESE-2, is exclusively expressed in a subset of epithelial cells with highest expression in glandular epithelium such as prostate, pancreas, salivary gland, and trachea. A potential role in branching morphogenesis is suggested, since ESE-3 transactivates the c-MET promoter via three high affinity binding sites. Additionally, ESE-3 binding to DNA sequences in the promoters of several glandular epithelium-specific genes suggests a role for ESE-3 in later stages of glandular epithelium differentiation. Although ESE-3 and ESE-1 bind with similar affinity to various Ets binding sites, ESE-3 and ESE-1 differ significantly in their ability to transactivate the promoters containing these sites. Our results support the notion that ESE-1, ESE-2, and ESE-3 represent a unique epithelium-specific subfamily of Ets factors that have critical but distinct functions in epithelial cell differentiation and proliferation.

PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.

Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Ets factor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.

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.

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).

Characterization of ESE-2, a novel ESE-1-related Ets transcription factor that is restricted to glandular epithelium and differentiated keratinocytes.

Epithelial cell differentiation is tightly controlled by distinct sets of transcription factors that regulate the expression of stage-specific genes. We recently isolated the first epithelium-specific Ets transcription factor (ESE-1). Here we describe the characterization of ESE-2, a second epithelium-restricted ESE-1-related Ets factor. Like ESE-1, ESE-2 is induced during keratinocyte differentiation. However, whereas ESE-1 is expressed in the majority of epithelial cell types, ESE-2 expression is restricted to differentiated keratinocytes and glandular epithelium such as salivary gland, prostate, mammary gland, and kidney. In contrast to ESE-1, full-length ESE-2 binds poorly to DNA due to the presence of a negative regulatory domain at the amino terminus. Furthermore, although ESE-1 and the amino-terminally deleted ESE-2 bind with similar affinity to the canonical E74 Ets site, ESE-2 and ESE-1 differ strikingly in their relative affinity toward binding sites in the c-MET and PSMA promoters. Similarly, ESE-1 and ESE-2 drastically differ in their ability to transactivate epithelium-specific promoters. Thus, ESE-2, but not ESE-1, transactivates the parotid gland-specific PSP promoter and the prostate-specific PSA promoter. In contrast, ESE-1 transactivates the keratinocyte-specific SPRR2A promoter Ets site and the prostate-specific PSMA promoter significantly better than ESE-2. Our results demonstrate the existence of a unique class of related epithelium-specific Ets factors with distinct functions in epithelial cell gene regulation.

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.

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.

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.

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.

A putative human zinc-finger gene (ZFPL1) on 11q13, highly conserved in the mouse and expressed in exocrine pancreas. The European Consortium on MEN 1.

In the process of identification of the multiple endocrine neoplasia type 1 gene, which was recently published, we isolated a novel gene in the 11q13 region. This gene (named ZFPL1, for zinc-finger protein-like 1) is expressed strongly in the exocrine pancreas as a 1.4-kb polyadenylated RNA encoding a putative protein of 310 amino acids. A mouse EST contig predicts an equally sized murine protein with 91% amino acid sequence identity to the human protein. No significant homology with known proteins could be found through database screening. However, zinc-finger-like domains and leucine-zipper-like motifs in the predicted ZFPL1 protein were identified, suggesting the presence of DNA-binding and dimerization domains possibly involved in transcription regulation. This notion is supported by the presence of a putative bipartite nuclear localization signal. This paper presents the full-length cDNA sequence for this gene, its genomic structure and chromosomal orientation, and expression studies by Northern blot hybridization and RNA in situ hybridization.

Expression and chromosomal localization of the Requiem gene.

Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.

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.

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.

Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1).

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing a conting, we have identified the location of the gene encoding the B56 beta subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations in the PP2A-B56 beta coding region, together with the 5' and 3' untranslated regions in six MEN1 patients. DNA sequence abnormalities were not identified and thus the PP2A-B56 beta gene is excluded as the candidate gene for MEN1. However, our precise localisation of PP2A-B56 beta to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this generich region.