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