The Role of Suppressor of Fused in Development and Tumorigenesis in the Mouse

Embryonic development is a process that involves a number of evolutionarily wellconserved signaling cascades, including the hedgehog pathway. Mutations in components of this pathway have been identified in certain developmental disorders, and in many different kinds of cancers. In fact, the most common cancer in the Western World, basal cell carcinoma (BCC) of the skin, is due to mutations that cause aberrantly activated hedgehog signaling. This thesis focuses on a protein known as Suppressor of fused (Sufu), which is an essential tumor suppressor within the hedgehog pathway. In PAPER I, we made the surprising observation that Sufu actually plays a fundamental role in the mammalian hedgehog signaling pathway, in contrast to its role in fruit flies and even zebrafish. In these organisms, Sufu plays an insignificant part in normal hedgehog signaling, since its absence only results in minor phenotypic changes. However, in the mouse, we showed that loss of Sufu leads to embryonic death in midgestation with the embryos exhibiting severe cephalic defects and an open neural tube. We also demonstrated that the Sufu loss-of-function phenotype was due to ligandindependent activation of the hedgehog signaling pathway. In humans, Gorlin syndrome is a rare developmental disorder that in the majority of cases is due to inactivating mutations in the gene that encodes the hedgehog receptor, PTCH1. This leads to overactivated hedgehog signaling, since PTCH1 is no longer able to inhibit the signal transducer, Smoothened (SMO). Gorlin syndrome involves an array of different developmental defects, but it also leads to increased tumor susceptibility, especially in the form of multiple BCCs. In PAPER I we discovered that mice, heterozygous for the Sufu gene, develop features of Gorlin syndrome, including a skin phenotype with BCC-like attributes, in addition to developmental aberrations in the form of jaw keratocysts. In addition, we showed that the extent of epidermal skin changes correlated with increased hedgehog pathway activation. The BCC-like lesions in Sufu+/- mice are reminiscent of basaloid follicular hamartomas (BFH), which are more benign lesions than BCCs. In PAPER II, the aim was to investigate whether the Sufu+/- skin lesions would develop into full-blown BCCs if Trp53 was knocked out simultaneously. Trp53 is a well-known tumor suppressor gene that can enhance hedgehog-driven tumors, and is often mutated in sporadic BCCs, sometimes in combination with PTCH1 mutations. We showed that Sufu+/- mice on a Trp53 null background develop medulloblastomas and rhabdomyosarcomas, which is consistent with previous reports. Surprisingly, however, the Sufu+/- skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype or proliferative capacity during the lifespan of the mice. This finding suggests a differential, tissue-specific sensitivity to Sufu and Trp53 gene loss, possibly linked to developmental phase and proliferative potential in specific tissues. In PAPER III, we studied developmental and differentiation processes in the absence of Sufu, using embryonic stem cells (ESCs) derived from Sufu-/- preimplantation embryos. Sufu-/- ESCs were found to express typical pluripotency markers, but the activity of the hedgehog pathway was increased only modestly compared to wild-type ESCs, as indicated by Gli1 target gene expression. The Sufu-/- ESCs formed embryoid bodies in vitro, which, in later stages, were smaller than their wild-type counterparts, suggesting a deficiency in proliferation. To test the differentiation capacity of the Sufu-/- ESCs in vivo, the cells were injected subcutaneously into nude mice to form teratomas. Teratomas from Sufu-/- ESCs developed at efficiencies and latencies equivalent to teratomas from wild-type ESCs, yet in stark contrast to wild-type, Sufu-/- teratomas were dominated by neuroectodermal tissues and were deficient in the mesodermal derivatives, cartilage and bone. These findings call attention to the central role played by Sufu in the hedgehog signaling pathway, and propose a function for Sufu in ectodermal-mesodermal cell fate decision. In a PRELIMINARY STUDY, we have generated conditional Sufu mutant mice with the aim of deleting Sufu in specific tissues at specific time-points. These studies are ongoing, and experiments to create mice with complete loss of Sufu in the K5 (basal cell) compartment of the skin have been initiated. In summary, the studies in this thesis highlight an essential role for Sufu in the hedgehog signaling pathway during development and tumorigenesis in mammals

Loss of <em>Trp53</em> promotes medulloblastoma development but not skin tumorigenensis in <em>Sufu</em> heterozyhous mutant mice

Basal cell carcinoma of the skin typically carries genetic alterations in components of the hedgehog (HH) signaling pathway. Previously, we generated a knockout mouse with a loss-of-function mutation in suppressor of fused (Sufu), an essential repressor of the pathway downstream of Hh ligand cell surface reception. Mice heterozygous for the mutated Sufu allele develop a skin phenotype that includes lesions similar to basaloid follicular hamartomas. The purpose of the current study was to test the possibility that the simultaneous loss of the tumor suppressor gene, Trp53, would aggravate the Sufu skin phenotype since Trp53 loss is known to enhance the growth of other Hh-driven tumors. Consistent with previous reports, medulloblastomas and rhabdomyosarcomas developed in Sufu(+/−);Trp53(−/−) mice. However, the characteristic Sufu(+/−) skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype or proliferative capacity of the basaloid lesions. This finding was both novel and intriguing and demonstrated a differential, tissue-specific sensitivity to Sufu and Trp53 tumor suppressor gene loss, which may be linked to developmental stage and the degree of proliferative activity in specific cell types