PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing and Up-Regulated in Basal Cell Carcinomas1

[CANCER RESEARCH 59, 787–792, February 15, 1999] Advances in Brief

PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing and Up-regulated in Basal Cell Carcinomas1

Peter G. Zaphiropoulos,2 Anne Birgitte Unde´n, Fahimeh Rahnama, Robert E. Hollingsworth,3 and Rune Toftgård2 Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum 141 57, Huddinge, Sweden [P. G. Z., A. B. U., F. R., R. T.], and Cancer Research, Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001 [R. E. H.]

Abstract appears to be involved in the SHH/PTCH cell signaling, having similar but still distinct properties than PTCH1. By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like Materials and Methods gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, cDNA and Gene Cloning and Radiation Hybrid Mapping. The RACE PTCH2 has been localized to chromosome 1p33-34, a region often lost in analysis was performed essentially as described before (10), using the Mara- a variety of tumors. Several alternatively spliced mRNA forms of PTCH2 thon kit (Promega). The primer sequences used for RACE are available on were identified, including transcripts lacking segments thought to be request. A BAC clone encompassing the PTCH2 gene was isolated using involved in sonic hedgehog binding and mRNAs with differentially de- primers corresponding to the 3Ј untranslated region of the cDNA. The genomic fined 3؅ terminal exons. In situ hybridization revealed high expression of organization was obtained by a combination of direct sequencing of the BAC PTCH2 transcripts in both familial and sporadic basal cell carcinomas in and by sequencing of PCR products generated with primers originating from similarity to what has been observed for PTCH1, suggesting a negative adjacent exons. The same 3Ј end primers used to isolate the BAC clone were regulation of PTCH2 by PTCH1. This finding tightly links PTCH2 with also used to screen the GENEBRIDGE radiation hybrid panel. the sonic hedgehog/PTCH signaling pathway, implying that PTCH2 has Tumor Samples. A total of 11 formalin-fixed paraffin-embedded BCCs related, but yet distinct, functions than PTCH1. derived from seven patients were obtained from the Department of Dermatol- ogy, Karolinska Hospital (Stockholm, Sweden). Five tumors were from Introduction NBCCS patients, two tumors were from a young patient with multiple BCCs (not classified as NBCCS), and four tumors were sporadic BCCs. All histo- PTCH 4 was recently identified as the gene responsible for the logical subtypes of BCCs were represented. In addition, samples from two TEs Gorlin’s syndrome, an autosomal dominant disorder characterized by were included. Both were sporadic tumors. The histological diagnoses were confirmed by an experienced dermatopathologist. multiple BCCs, medulloblastomas, and ovarian fibromas, as well as In Situ Hybridization. Preparation of PTCH2-mRNA probes and in situ numerous developmental anomalies (1, 2). PTCH codes for a mem- hybridization were performed as described previously (9). Briefly, two human brane receptor of the autocatalytically cleaved (protein-spliced), ami- PTCH2 cDNA fragments corresponding to positions 218–437 and 838–920, no-terminal domain of SHH (3, 4). In the nonsignaling state, PTCH is cloned into pGEM5, were used. Tumor sections were treated with proteinase thought to inhibit the constitutive signaling of another membrane K and washed in 0.1 M triethanolamine buffer containing 0.25% acetic anhy- protein, SMO, however, binding of SHH to PTCH relieves this dride. Sections were hybridized with 2.5 ϫ 106 cpm of each of the two labeled inhibition (5). This cascade of signaling events, best characterized in antisense or sense probes at 55°C. Autoradiography was performed for 2 Drosophila, also involves a number of intracellular components in- weeks. Immunohistochemistry. A monoclonal mouse antibody directed against cluding fused (a serine threonine kinase), suppressor of fused, costal human Ki-67 was used (Immunotech). Sections from each tumor sample were 2, and cubitus interruptus (6). The latter is a transcription factor that incubated with the antibody for 1 h and processed using a standard avidin- positively regulates the expression of target genes that also include biotin immunoperioxidase/diaminobenzidine detection system. PTCH itself. Mutations in the PTCH gene have been identified in both sporadic and familial BCCs (7, 8). The lack of the normal PTCH Results and Discussion protein in these cells allows the constitutive signaling of SMO to To identify additional components of the PTCH/SHH cascade of occur, resulting in the accumulation of mutant PTCH mRNAs (9). In signaling events, the Incyte LifeSeqTM database was searched using this study, we describe the cloning and characterization, including PTCH sequences. In addition to clones representing the PTCH cDNA, differentially spliced mRNA forms, of a related gene, PTCH2,5 that two nearly identical cDNAs were identified from the parotid gland and the colon, which contained sequences similar to, but distinct from, Received 11/30/98; accepted 12/31/98. the 3Ј end of PTCH. By 5Ј RACE analysis using fetal brain cDNAs, 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 additional sequence information from these transcripts (termed 18 U.S.C. Section 1734 solely to indicate this fact. PTCH2) and, corresponding to a full-length cDNA, was obtained (Fig. 1 Supported by the Swedish Cancer Fund, the Welander-Finsen Foundation, the Swedish Radiation Protection Institute, the Swedish Children Cancer Fund, and the 1A). PTCH2 is 57% identical to PTCH1, with a significantly variable Karolinska Institute. region present between the transmembrane domains 6 and 7, and 91% 2 To whom requests for reprints should be addressed, at Department of Bioscience, identical to the recently published mouse Ptch2 sequence (11). In Center for Nutrition and Toxicology, Karolinska Institute, Novum 141 57, Huddinge, Sweden. Phone: 46-8-6089151 (P. G. Z.) or 46-8-6089152 (R. T.); Fax: 46-8-6081501; similarity with the mouse gene, PTCH2 lacks the COOH-terminal E-mail: [email protected] or [email protected]. extension present in human, mouse, and chicken PTCH1 (12, 13). 3 Present address: Genomic Sciences, Glaxo Wellcome Inc., Research Triangle Park, However, the human PTCH2 cDNA terminates 36 amino acids earlier NC 27709. 4 The abbreviations used are: PTCH, human patched; BCC, basal cell carcinoma; SHH, than the mouse Ptch2 sequence. Characterization of the PTCH2 gene sonic hedgehog; SMO, smoothened; TE, trichoepithelioma; RACE, rapid amplification of revealed 22 coding exons and conservation of the intron-exon junc- cDNA ends; NBCCS, nevoid basal cell carcinoma syndrome. 5 The PTCH2 sequence has been submitted to the GenBank (accession number tions relative to PTCH1, except for the last intron (Fig. 1B). More- AF119569). over, when 3Ј RACE was performed from fetal brain, an alternate 787

Fig. 1. A, amino acid sequence comparison of the human PTCH2 (top lines) and PTCH1 sequences. Vertical lines, identical amino acids; dots, similar amino acids. The PTCH2 sequence presented is composed of the original cDNA clones and of the products of the 5Ј RACE analysis. B, intron-exon organization of the PTCH2 gene. The intron-exon borders are shown, with small letters representing intronic sequences and capital letters representing exonic sequences. C, schematic representation of the alternative splicing events that result in different COOH termini. In the parotid gland and the colon, the penultimate and the last exon are canonically joined together. In fetal brain, however, the penultimate exon with part of the 3Ј intron functions as the terminal exon. The intronic sequence is shown by small letters, with the flanking exonic shown by capital letters. Above the nucleotide sequence, the deduced amino acid sequence is shown, and below is the corresponding sequence of mouse Ptch2. The conserved intronic dinucleotides are shown by bold letters, and the termination signals are indicated by *. Note the absence of conservation of the position of the termination codons between the mouse and human PTCH2 sequences. The putative polyadenylation signals are also shown in this diagram. The genomic organization was obtained by analyzing a BAC clone encompassing the PTCH2 gene. D, schematic representation of the different variations of spliced transcripts encompassing exon 1 and exon 2 sequences. The canonical exons 1 and 2 are shown by boxes, and the intron between them is shown by a solid line. The GT and AG dinucleotides spanning the sequences that are used as introns in individual transcripts are indicated by small letters. G, genomic structure, derived from sequencing segments of a BAC clone encompassing the PTCH2 gene; C, canonical transcript; A, transcript A (the skipped exons 9 and 10 of this product are not shown in the diagram); B, transcript B. 788

Fig. 1. C, D, Continued.

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Fig. 2. A, a dark-field photomicrograph of a BCC tumor hybridized with 35S-labeled antisense probe showing abundant signal for PTCH1 mRNA (light grains) in all BCC tumor cells. B, PTCH2 mRNA overexpression in BCC is in contrast mainly expressed in the basaloid cells in the periphery of the tumor nests (arrow). C, another BCC showing a strong PTCH2 mRNA signal in the periphery of the tumor nest (Tu), whereas no signal is detected in epidermis (Ep). D, sections of the same tumor (C) hybridized with the PTCH2 sense probe showed no signal. E, immunoreactivity for Ki-67 (brown precipitate) is seen in the periphery in the cells that showed strong up-regulation of PTCH2 mRNA. F, tumor nests under high power magnification demonstrate abundant PTCH2 mRNA signal (black grains) in the dark basaloid tumor cells and lower signal in the center (arrow). Bars (A-E), 24 mm and6mm(F).

COOH-terminal region was identified. This had a high structural transcripts were also identified by the RACE analysis (Fig. 1D). similarity with the mouse Ptch2 COOH-terminal sequence and Transcript A lacks the sequence that corresponds to exons 9 and 10 originates from the genomic region that links the last two exons of of PTCH1, with the open reading frame being retained at the exon PTCH2 (Fig. 1C). Therefore, in these alternatively spliced tran- 8 to exon 11 junction. Exons 9 and 10 code for the last part of the scripts, the penultimate exon with a segment of the contiguous 3Ј first extracellular loop and for transmembrane domains 2 and 3 in intron serves as the terminal exon. Furthermore, the human and the putative structure of the PTCH1 protein. Furthermore, this mouse transcripts differed in the position of the termination signals transcript also lacks a 5Ј segment of the canonical exon 2, due to (the human sequence is 21 amino acids longer), suggesting a the use of an alternative 3Ј splice site present in this exon, with the nonconserved, species-specific function of this alternate COOH- open reading frame being maintained. The functional consequence terminal domain. The finding of two possible COOH-terminal of this alternative splicing is not yet known, but it is interesting to regions for PTCH2 is intriguing and implies a role of this phe- note that the extracellular loops in PTCH1 are presumed to be nomenon in modulating signaling. Additional alternatively spliced involved in binding of the ligand SHH (3, 4) and that insertion of 790

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A NOVEL PTCH GENE a neo-cassette in intron 9 of the mouse PTCH1 gene is associated Gorlin’s syndrome. However, whether PTCH2 may block the consti- with a severe phenotype (14). Furthermore, exons 9 and 10 encode tutive signaling of SMO or could act as an additional SHH receptor, part of a putative sterol sensing domain (15), also found in PTCH1, possibly dependent on alternative splicing, remains as the subject of and which has recently been implicated in mediating the potent further experimentation. modulating effect of cholesterol on SHH/PTCH signaling (16). Thus, if PTCH2 also serves as a receptor for SHH and/or related factors, the receptor form lacking exons 9 and 10 may show altered Acknowledgments signaling properties. Transcript B contains additional sequences We thank Karima Raza and Lotta Malmqvist for assistance in the sequenc- Ј between canonical exons 1 and 2 that originate from the 5 end of ing PTCH2 and Bengt Sandstedt, MD., Ph.D., for help with the Ki-67 immu- intron 1. The open reading frame that includes the initiator methi- nostaining. onine of exon 1 is not maintained in this transcript, suggesting that, if this transcript is functional, either the methionine in exon 2 or nonmethionine codons are used to produce a protein product, in References similarity to what has been proposed for the alternative spliced 1. Hahn, H., Wicking, C., Zaphiropoulos, P. G., Gailani, M. R., Shanley, S., products of human PTCH1 (1). Chidambaram, A., Vorechovsky, I., Holmberg, E., Undeń, A. B., Gillies, S., Negus, By radiation hybrid mapping, the PTCH2 gene was localized to K., Smyth, I., Pressman, C., Leffell, D. J., Gerrard, B., Goldstein, A. M., Dean, M., Toftgård, R., Chenevix-Trench, G., Wainwright, B., and Bale, A. E. Mutations of the the chromosome 1p33-1p34 region, flanked by microsatellite human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. markers D1S197 and D1S443. Interestingly, several genetic dis- Cell, 85: 841–851, 1996. eases have been mapped in this 2cM chromosomal segment, in- 2. Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M., Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr., and Scott, M. P. Human cluding paroxysmal choreoathetosis (17) and a form of hereditary homolog of patched, a candidate gene for the basal cell nevus syndrome. Science congenital ptosis (18). The presence of PTCH2 transcripts in (Washington DC), 272: 1668–1671, 1996. several ocular tissues (19) provides additional evidence that this 3. Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M., and Tabin, C. J. Biochemical evidence that patched is the Hedgehog receptor. Nature (Lond.), gene might be a good candidate for the above mentioned hereditary 384: 176–179, 1996. eye disorder. In addition, PTCH2 is also located within the putative 4. Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., candidate region for the cancer predisposition locus hMOM1 (20), Scott, M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F., and Rosenthal, A. The tumour-suppressor gene patched encodes a and this region is, furthermore, often lost in several tumor types candidate receptor for Sonic hedgehog. Nature (Lond.), 384: 129–134, 1996. including neuroblastomas and colorectal tumors (21–23). Whether 5. Goodrich, L. V., Milenkovic, L., Higgins, K. M., and Scott, M. P. Altered neural cell or not PTCH2, in similarity to PTCH1, is a tumor suppressor gene fates and medulloblastoma in mouse patched mutants. Science (Washington DC), 277: 1109–1113, 1997. is presently under investigation. 6. Ruiz i Altaba, A. Catching a glimpse of Hedgehog. Cell, 90: 193–196, 1997. The mouse and zebrafish homologues of PTCH2 have been re- 7. Gailani, M. R., Ståhle-Ba¨ckdahl, M., Leffell, D. J., Glynn, M., Zaphiropoulos, P. G., ported to be expressed in a partly overlapping pattern with PTCH1 Pressman, C., Undeń, A. B., Dean, M., Brash, D. E., Bale, A. E., and Toftgård, R. The during embryonic development and to be induced by SHH (11, 24), role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat. Genet., 14: 78–81, 1996. implicating a role in this signaling pathway. We were, with this 8. Undeń, B. A., Holmberg, E., Lundh-Rozell, B., Ståhle-Ba¨ckdahl, M., Zaphiropoulos, background, interested to analyze the expression of PTCH2 in BCCs P. G., Toftgård, R., and Vorechovsky, I. Mutations in the human homologue of and TEs, which show consistent upregulation of PTCH1 in all tumor Drosophila patched (PTCH) in basal cell carcinomas and the Gorlin’s syndrome: different in vivo mechanisms of PTCH inactivation. Cancer Res., 56: 4562–4565, cells (9, 25). By in situ hybridization analysis, a positive signal for 1996. PTCH2 mRNA was seen in all the 11 BCCs and the 2 TEs examined. 9. Undeń, B. A., Zaphiropoulos, P. G., Bruce, K., Toftgård, R., and Ståhle-Ba¨ckdahl, The signal was seen exclusively in the tumor cells in both tumor types M. Human patched (PTCH) mRNA is overexpressed consistently in tumor cells of both familial and sporadic basal cell carcinoma. Cancer Res., 57: 2336–2340, and was consistently stronger in the palisading peripheral cells of the 1997. tumor nests (Fig. 2). These cells also showed a positive immuno- 10. Zaphiropoulos, P. G., and Toftgård, R. cDNA cloning of a novel WD repeat staining for the cell proliferation marker, Ki-67. Moreover, no or only protein mapping to the 9q22.3 chromosomal region. DNA Cell Biol., 15: 1049– 6 1056, 1996. weak signal could be detected in normal epidermis or in hair follicles. 11. Motoyama, J., Takabatake, T., Takeshima, K., and Hui, C. Ptch2, a second mouse It is interesting to note that the prominent PTCH2 expression was Patched gene is co-expressed with Sonic hedgehog. Nat. Genet., 18: 104–106, observed both in familial (NBCCS, n ϭ 5) and sporadic (n ϭ 6) 1998. 12. Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A., and Scott, M. P. tumors. One of the BCCs analyzed was from a NBCCS patient (Fig. Conservation of the hedgehog/patched signaling pathway from flies to mice: induc- 2, C-F), with a known 4-bp germline deletion in exon 3 in the PTCH1 tion of a mouse patched gene by Hedgehog. Genes Dev., 10: 301–312, 1996. gene, which results in a premature stop and a truncated protein.6 13. Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V., and Tabin, C. J. Conser- Furthermore, one of the TEs was earlier shown to contain a large vation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development, 122: 1225–1233, 1996. deletion in exon 20 that removes the COOH-terminal part of the 12th 14. Hahn, H., Wojnowski, L., Zimmer, A. M., Hall, J., Miller, G., and Zimmer, A. transmembrane domain of the PTCH1 gene (25). Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin’s The finding that in BCCs having frequent mutations in the PTCH1 syndrome. Nat. Med., 4: 619–622, 1998. 15. Osborne, T. F., and Rosenfeld, J. M. Related membrane domains in proteins of sterol gene the expression of the PTCH2 mRNAs is up-regulated tightly sensing and cell signaling provide a glimpse of treasures still buried within the links PTCH2 with the PTCH/SHH cascade of signaling events. It is, dynamic realm of intracellular metabolic regulation. Curr. Opin. Lipidol., 9: 137–140, therefore, likely that PTCH2 represents a target gene of this pathway, 1998. 16. Cooper, M. K., Porter, J. A., Young, K. E., and Beachy, P. A. Teratogen-mediated which is under the negative regulation of PTCH1, precisely as PTCH1 inhibition of target tissue response to Shh signaling. Science (Washington DC), 280: itself. Moreover, this observation strongly suggests that PTCH2 has 1603–1607, 1998. functions distinct from PTCH1 since up-regulation of PTCH2 expres- 17. Auburger, G., Ratzlaff, T., Lunkes, A., Nelles, H. W., Leube, B., Binkofski, F., Kugel, H., Heindel, W., Seitz, R., Benecke, R., Witte, O. W., and Voit, T. A gene for sion seems unable to compensate for inactive PTCH1 protein. This autosomal dominant paroxysmal choreoathetosis/spasticity (CSE) maps to the vicin- conclusion is also supported by the early embryonic lethality seen in ity of a potassium channel gene cluster on chromosome 1p, probably within 2 cM PTCH1 (Ϫ/Ϫ) mice (5, 14) and the lack of genetic heterogeneity in between D1S443 and D1S197. Genomics, 31: 90–94, 1996. 18. Engle, E. C., Castro, A. E., Macy, M. E., Knoll, J. H., and Beggs, A. H. A gene for isolated congenital ptosis maps to a 3-cM region within 1p32–p34.1. Am. J. Hum. 6 Unpublished observations. Genet., 60: 1150–1157, 1997. 791

19. Takabatake, T., Ogawa, M., Takahashi, T. C., Mizuno, M., Okamoto, M., and 23. Lothe, R. A., Andersen, S. N., Hofstad, B., Meling, G. I., Peltomaki, P., Heim, S., Takeshima, K. Hedgehog and patched gene expression in adult ocular tissues. FEBS Brogger, A., Vatn, M., Rognum, T. O., and Borresen, A. L. Deletion of 1p loci and Lett., 410: 485–489, 1997. microsatellite instability in colorectal polyps. Genes Chromosomes Cancer, 14: 20. Dobbie, Z., Heinimann, K., Bishop, D. T., Muller, H., and Scott, R. J. Identification 182–188, 1995. of a modifier gene locus on chromosome 1p35–36 in familial adenomatous polyposis. 24. Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V., Johnson, R. L., Scott, Hum. Genet., 99: 653–657, 1997. M. P., and Ingham, P. W. Spatial regulation of a zebrafish patched homologue reflects 21. Mertens, F., Johansson, B., Hoglund, M., and Mitelman, F. Chromosomal imbalance the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning. maps of malignant solid tumors: a cytogenetic survey of 3185 neoplasms. Cancer Development, 122: 2835–2846, 1996. Res., 57: 2765–2780, 1997. 25. Vorechovsky, I., Unde´n, A. B., Sandstedt, B., Toftgård, R., and Ståhle-Ba¨ckdahl, M. 22. Ohtsu, K., Hiyama, E., Ichikawa, T., Matsuura, Y., and Yokoyama, T. Clinical Trichoepitheliomas contain somatic mutations in the overexpressed PTCH gene: investigation of neuroblastoma with partial deletion in the short arm of chromosome support for a gatekeeper mechanism in skin tumorigenesis. Cancer Res., 57: 4677– 1. Clin. Cancer Res., 3: 1221–1228, 1997. 4681, 1997.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing andUp-regulated in Basal Cell Carcinomas

Peter G. Zaphiropoulos, Anne Birgitte Undén, Fahimeh Rahnama, et al.

Cancer Res 1999;59:787-792.

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