Basal Cell Carcinomas in Mice Overexpressing Sonic Hedgehog Anthony E. Oro, Kay M. Higgins, Zhilan Hu, Jeannette M. Bonifas, Ervin H. Epstein Jr.,* Matthew P. Scott* Mutations in the tumor suppressor gene PATCHED (PTC) are found in human patients with the basal cell nevus syndrome, a disease causing developmental defects and tumors, including basal cell carcinomas. Gene regulatory relationships defined in the fruit fly Drosophila suggest that overproduction of Sonic hedgehog (SHH), the ligand for PTC, will mimic loss of ptc function. It is shown here that transgenic mice overexpressing SHH in the skin develop many features of basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas in mice. These data suggest that SHH may have a role in human tumorigenesis. A large body of evidence supports the idea that multiple genetic events are required to transform normal epithelium into benign growths and then into metastatic tumors (1). Some types of tumors rarely show com- plete progression: For example, basal cell carcinomas (BCCs) of the skin—the most common tumors in Caucasians, with about 750,000 new cases annually in the United States—are generally only locally invasive (2). The lack of a mouse model of BCCs and the difficulty in culturing human BCCs has slowed progress in understanding the mechanisms underlying BCC biology. Basal cell nevus syndrome (BCNS) is an autosomal dominant disease characterized by developmental defects and a predisposi- tion to certain tumors (3). The most com- mon morphologic abnormalities are skeletal defects such as polydactyly, jaw and rib defects, and spina bifida; the most common tumors are BCCs, medulloblastomas, and meningiomas. The defective gene is ptc (4), a gene on chromosome 9q, first identified in Drosophila as a regulator of embryonic pat- tern formation. Numerous sporadic BCCs also have 9q loss and ptc mutations, suggest- ing that many BCCs unrelated to BCNS arise from somatic damage to both copies of ptc (4, 5). The ptc gene encodes a transmembrane receptor that represses transcription of genes encoding transforming growth fac- tor– and Wnt class signaling proteins and ptc itself (6). One vertebrate PTC ligand is the secreted protein SHH, which binds to PTC in cultured cells and frog oocytes. The fly homolog of SHH, Hedgehog (HH), is believed to inactivate PTC function, sug- gesting that HH proteins induce target gene transcription by inactivating their recep- tor’s function. The COOH-terminal part of SHH is an autoprotease and cholesterol transferase that cleaves the SHH precursor into two fragments and adds a cholesterol moiety to the NH 2 -terminal fragment (6). The latter fragment is sufficient for all known signal- ing events and contains a zinc hydrolase- like domain that may act as a peptidase (6, 7). Thus, in addition to binding to PTC, SHH may cleave an unknown target mol- ecule, although no catalytic activity has yet been detected. In Drosophila, ptc re- presses its target genes except where ptc function is inactivated by Hh, and this relationship appears to be conserved in vertebrates. Excess Hh function has an effect similar to loss of ptc function (8). This genetic relationship means that over- expression of Shh in mouse skin might mimic the loss of ptc function seen in human BCCs. In normal mice, Shh and ptc RNA accu- mulate in follicular but not interfollicular skin. Initial expression of Shh and ptc in skin occurs in Hardy stage 1 hair follicles. In 14.5-day postcoital (dpc) skin, Shh RNA accumulates at regularly spaced intervals in the ectoderm. Each spot of Shh signal over- lies the mesenchymal condensation of a presumptive follicle (9) (Fig. 1A). High levels of ptc RNA accumulate in each un- derlying mesenchymal condensation and at slightly lower levels in the Shh-expressing ectodermal cells (Fig. 1B), presumably be- cause of induction of ptc transcription by SHH (10). To examine in vivo the effect of excess SHH signaling, we generated transgenic mice that overexpress SHH specifically in the skin. We fused Shh to the keratin 14 (K14) promoter (Fig. 1I) (11), which drives expression as early as 9.5 dpc in the ecto- derm and at later stages in both the follic- ular and interfollicular epithelium (12). In total, 26 transgenic mice derived from pro- nuclear injection were examined as embry- os or neonates; lines could not be estab- lished because of perinatal lethality. In transgenic embryos, high levels of Shh RNA and ptc RNA accumulated in the basal layer of the epidermis, both in the follicular and the interfollicular epithelium (Fig. 1, C and D). The heightened ptc expression con- firmed that functional SHH was present and capable of inducing the ptc target gene in epidermal cells. The ptc transcripts were also present in the mesenchyme underlying the ectoderm of transgenics, presumably re- sulting from movement of SHH into these cells. The K14-Shh transgenic mice exhibited skeletal and skin abnormalities reminis- cent of those seen in BCNS. The most frequent abnormality was polydactyly of both the fore- and hind limbs, some of which had eight digits (Fig. 1E). Each digit looked similar to the normal central digits, as in the chick talpid 3 mutant (13). Distal phalanges were often missing, giv- ing rise to a shortened but wider limb. Distal cartilage bifurcations, ectopic sites of cartilage formation between the digits, and a distal rim of persistent ossification were apparent. Spina bifida, a failure to close the neural tube, was also frequently observed in the transgenics. This defect always affected the caudal portion of the spine and, in severe mutants, extended to the thoracic spine. Skeletal preparations revealed that spinal processes that normal- ly enclose the spinal cord failed to form dorsally (Fig. 1F) (14). These effects on skeletal development suggest that SHH penetrates internal tissues, as does the mesenchymal ectopic ptc expression. The K14-Shh transgenic mice had mul- tiple BCC-like epidermal proliferations throughout their skin surface after only the first few days of skin development. Dead perinatal embryos invariably had erosions that destroyed much of the skin surface. The skin lacked normal folds and was trans- lucent and friable (Fig. 1H). Skin histology revealed massive proliferations of cells asso- ciated with primordial invaginating hair follicles, which were hyperchromatic but cytologically normal. At 18.5 dpc, the epi- dermal proliferations often involved most of the epidermal surface (Fig. 2, B and C). In mildly affected embryos, one or two epider- mal growths were interspersed with six to eight follicles that appeared normal (Fig. 2D). In human BCCs, epidermal cells pro- A. E. Oro, Howard Hughes Medical Institute, Depart- ments of Dermatology, Developmental Biology, and Ge- netics, Stanford University School of Medicine, Stanford, CA 94305 –5427, USA. K. M. Higgins and M. P. Scott, Howard Hughes Medical Institute, Departments of Developmental Biology and Ge- netics, Stanford University School of Medicine, Stanford, CA 94305 –5427, USA. Z. Hu, J. M. Bonifas, E. H. Epstein Jr., Department of Dermatology, San Francisco General Hospital, University of California, San Francisco, CA 94110, USA. * To whom correspondence should be addressed. E-mail addresses: scott@cmgm.stanford.edu, ehepstein@orca. ucsf.edu REPORTS www.sciencemag.org  SCIENCE  VOL. 276  2 MAY 1997 817