Oncogene (2001) 20, 6707 ± 6717 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc LAPSER1: a novel candidate tumor suppressor gene from 10q24.3 Yofre Cabeza-Arvelaiz1, Timothy C Thompson2, Jorge L Sepulveda3 and A Craig Chinault*,1 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, TX 77030, USA; 2Department of Urology, Baylor College of Medicine, Houston, Texas, TX 77030, USA; 3Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, PA 15261, USA Numerous LOH and mutation analysis studies in Transfer of these portions of each chromosome into dierent tumor tissues, including prostate, indicate that cancer cells has provided further evidence indicating there are multiple tumor suppressor genes (TSGs) that these regions harbor genes that suppress tumor- present within the human chromosome 8p21 ± 22 and igenicity (Ichikawa et al., 1994; Murakami et al., 1996). 10q23 ± 24 regions. Recently, we showed that LZTS1 (or Several candidate prostate cancer genes have been FEZ1), a putative TSG located on 8p22, has the isolated from 8p21 ± 22, including PRLTS (Fujiwara et potential to function as a cell growth modulator. We al., 1995), N33 (MacGrogan et al., 1996), and FEZ1 report here the cloning, gene organization, cDNA (Ishii et al., 1999). The expression of the latter, which sequence characterization and expression analysis of has now been designated as the LZTS1 (leucine zipper, LAPSER1,anLZTS1-related gene. This gene maps putative tumor suppressor 1) gene, has been found to within a subregion of human chromosome 10q24.3 that be altered in many tumors and cell lines including has been reported to be deleted in various cancers, esophageal, breast and prostate (Ishii et al., 1999). We including prostate tumors, as frequently as the neighbor- have also recently demonstrated this gene's ability to ing PTEN locus. The complete LAPSER1 cDNA in¯uence cellular growth properties (Cabeza-Arvelaiz et sequence encodes a predicted protein containing various al., 2001), which, together with the previous results, domains resembling those typically found in transcription make it an excellent candidate for a TSG. factors (P-Box, Q-rich and multiple leucine zippers). The tumor suppressor gene PTEN, whose loss of LAPSER1 is expressed at the highest levels in normal function has been shown or suggested in multiple types prostate and testis, where multiple isoforms are seen, of cancer, including Cowden disease, Bannayan- some of which are either undetectable or dierentially Zonana syndrome, and glioma, kidney, breast and expressed in some prostate tumor tissues and cell lines. prostate tumors, is located at 10q23.3 (Li et al., 1997; Over-expression of LAPSER1 cDNA strongly inhibited Steck et al., 1997; Teng et al., 1997; Vlietstra et al., cell growth and colony-forming eciencies of most 1998). Actually, multiple neoplasms with recurrent cancer cells assessed. Together these data suggest that chromosomal aberrations in this region have been LAPSER1 is another gene involved in the regulation of described by the NCI Cancer Genome Anatomy cell growth whose loss of function may contribute to the Project (http://www.ncbi.nlm.nih.gov/CGAP/mitel- development of cancer. Oncogene (2001) 20, 6707 ± sum.cgi). Cancers with the unbalanced chromosomal 6717. abnormality del(10)(q24) include acute lymphoblastic and myeloid leukemias, astrocytomas, adenocarcinoma Keywords: prostate cancer; tumor suppressor; LZTS1; of various tissues (breast, large intestine, ovary, LAPSER1 prostate and stomach), bladder transitional cell carcinoma, undierentiated carcinoma of the prostate, chronic lymphoproliferative disorder, testis germ cell Introduction tumor, mesothelioma, non-Hodgkin's lymphoma, and uterus leiomyomas. Cancers with balanced chromoso- Loss of heterozygosity (LOH) and homozygous mal abnormalities that involve this region include acute deletions at human chromosomal regions 8p21 ± 22 lymphoblastic leukemias (t(10;14)(q24;q11), non- and 10q23 ± 24 are frequently found in prostate Hodgkin's lymphoma (t(10;14)(q24;q11), and adenocarcinomas, as well as other cancer tumors, and (t(10;14)(q24;q32), and thyroid adenocarcinoma suggest that multiple TSGs are present in each of these ((t(10;19)(q24;q13). This great variety of cancers with locations (Bova et al., 1996; Cairns et al., 1997; Carter apparent relevant genes in this region support the et al., 1990; Gray et al., 1995; Ishii et al., 1999; possibility that there may also be additional TSGs Ittmann, 1996; Kagan et al., 1995; Kim et al., 1998; Li located there that have not yet been characterized. et al., 1997; Petersen et al., 1998; Whang et al., 1998). We report here the cloning, cDNA sequencing, gene organization and predicted protein structure of LAPSER1, a novel LZTS1-related candidate TSG *Correspondence: AC Chinault; E-mail: [email protected] located on human chromosome 10q24.3 near the Received 10 May 2001; revised 20 July 2001; accepted 26 July 2001 PTEN locus. Expression analysis revealed that this The LAPSER1 gene Y Cabeza-Arvelaiz et al 6708 gene is expressed in most normal tissues, with the highest abundance found in prostate and testis. In addition, expression analysis revealed that multiple isoforms of this gene are dierentially expressed in dierent cell lines and tissues. Analysis of the eects of over-expression of LAPSER1 on colony formation and cell growth of several cancer cell lines suggests that LAPSER1 is another attractive cell growth control gene whose loss of function may be of relevance to the development of prostate cancer. Results Identification and mapping of the LAPSER1 gene LZTS1 (also known as FEZ1) has been suggested as a candidate TSG on the basis of its frequent deletion in various cancers (Ishii et al., 1999) and its recent functional identi®cation as a cell growth suppressing gene on 8p22 (Cabeza-Arvelaiz et al., 2001). When BLAST searches were performed with the LZTS1 gene sequence against the GenBank databases, we identi®ed three other related human genes of unknown function, as shown in the dendogram in Figure 1a. The sequence with highest overall sequence identity to LZTS1 corresponded to a gene region on contig AL133215 that we have provisionally designated as LAPSER1 (for its high content of the amino acids leucine (L), alanine (A), proline (P), serine (S), glutamic acid (E), and arginine (R)). This region has been mapped by FISH analysis to human chromosome 10q24.1 ± 24.33 (http://webace.sanger.ac.uk). To more precisely place the LAPSER1 gene relative to the PTEN locus, we compiled sequencing and mapping data from several sources, as described in Materials and methods, to construct a physical map encompassing 10q24, as shown in Figure 1b. Cloning and organization of the LAPSER1 gene To study the LAPSER1 gene and to explore its possible involvement in the regulation of cell growth, we cloned its complete cDNA sequence, using a RT ± PCR based approach with normal human prostate and testis RNA. An initial nested PCR ampli®cation performed with gene speci®c primers that ¯anked the presumed LAPSER1 coding region Figure 1 (a) Phylogenetic analysis of LAPSER1 and related (inferred by comparison to the LZTS1 cDNA) human protein sequences. The dendogram depicting the relation- ship between the indicated protein sequences was compiled using yielded one major and two minor transcription ClustalW analysis from the Mac Vector 3.5 software package. products of approximately 2, 1.7, and 1.35 Kb, The phylogenetic distances between the sequences are indicated, designated I, II, and III, respectively, in Figure 2a. where a value of 0.1 corresponds to a dierence of 10% between PCR re-ampli®cation of the three major RT ± PCR the two sequences. (b) Chromosomal location of the LAPSER1 products with internal primer sequences from exons gene. An ideogram of chromosome 10 is shown along with the physical and transcript map of the 10q23.3 ± 24.3 sub-region 2, 3 or 4 paired with an exon 5 primer (¯anking the spanning *22 Mb between the PTEN and MXI1 genes. The coding sequence) suggested that the minor transcripts locations of two of the most frequently deleted segments of this were generated by alternative exon splicing (Figure sub-region in prostate cancer, based on analysis with the markers 2b). Transcript I contained the expected sequences shown, are enclosed by open boxes. The loci of several known genes that have been mapped to this sub-region are shown. The from all coding exons tested while transcripts II and estimated distances from the p-telomore are indicated in III lacked exon 4 (258 bp) and exon 3 (660 bp), megabases (Mb), based on the scale of the Human Genome respectively. Browser website (http://genome.ucsc.edu) Oncogene The LAPSER1 gene Y Cabeza-Arvelaiz et al 6709 Figure 2 Cloning and genomic organization of the LAPSER1 gene. (a) RT ± PCR products of alternatively spliced transcripts of LAPSER1 using gene speci®c primers ¯anking the coding sequence are indicated by arrowheads on the left. (b) PCR analysis of the LAPSER1 mRNA isoforms detected in a, using primer sets designed to amplify coding sequences from either exon 2, exon 3 or exons 4 ± 5 (after the stop codon). The sizes of the PCR products in kilobases are indicated with arrows on the left. In (a) and (b), lanes marked M contain the size markers (100 bp ladder). (c) Schematic representation of the assembly of the full-length LAPSER1 cDNA sequence. The coding region (represented by black boxes), was obtained by sequencing the RT ± PCR-derived transcript I. Sequences from the indicated EST clones (represented by hatched bars and identi®ed by the GenBank accession number), were used to assemble a composite of the complete sequence of the LAPSER1 cDNA. Intron sequences are shown as dashed lines. (d) Comparison of the organization of the human LAPSER1 and LZTS1 genes. The coding region (indicated as closed boxes) is contained within exons 2 (451 bp), 3 (660 bp), 4 (258 bp), and 5 (1320 bp) of the LAPSER1 gene; thin connecting lines to the LZTS1 schematic indicate the orthologous exons.