Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.

The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449–resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein–coupled receptor can serve as a mechanism of drug resistance in human cancer.

Half a century ago the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal-distal (P-D) axis, but how these signals influence limb patterning is still much debated 1,2 . FGF gene family members are key 4 , with Fgf4, Fgf8, Fgf9, and Fgf17 expressed specifically in the mouse AER 5 . Here, we demonstrate that limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype 6,7 , but when we also removed different combinations of the other AER-FGF genes we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution each FGF can make to total AER-FGF signal. Analysis of the compound mutant limb buds revealed that in addition to sustaining cell survival, AERFGFs regulate P-D patterning gene expression during early limb bud development, thus providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signaling is permissive rather than instructive for limb patterning. We discuss how a twosignal model for P-D patterning can be integrated with the concept of early specification to explain the genetic data presented here. KeywordsAER; apical ectodermal ridge; cell survival; FGF4; FGF8; FGF9; FGF17; limb development; proximal-distal patterning Fgf8 is expressed in prospective AER cells of the nascent limb bud and subsequently throughout the AER until it regresses 8 , whereas Fgf4, Fgf9, and Fgf17 expression commences after the AER is formed, is restricted to the posterior AER, and ceases at least a day before AER regression 5 (Fig. 1a). When AER-FGFs are individually eliminated, only loss of Fgf8 function perturbs skeletal patterning 5-7,9-11 . The other AER-FGFs have been proposed to be essential, but functionally redundant components of a positive-feedback loop between the AER and the patterning center in posterior limb bud mesenchyme that produces Sonic hedgehog (SHH) 5,12,13 . We tested this hypothesis by deleting Fgf4 via Cre-mediated recombination in the AER of embryos homozygous for Fgf9 and Fgf17 null alleles 10,11 (hereafter referred to as F4;9,17-triple knock-out [TKO] mutants; Fig. 1b). Because Fgf4 deletion occurs before Fgf4 expression normally commences 5 (see Fig. 1a), the F4;9,17-TKO limb buds do not produce FGF4, or FGF9 or FGF17. Nevertheless, in F4;9,, the three classically defined limb segments, stylopod (S, upper arm or leg), zeugopod (Z, lower arm or leg), and autopod (A, wrist/hand or ankle/foot), were essentially normally patterned (Fig. 1c) Consistent with this observation, Shh expression appeared normal in F4;9,17-TKO limb buds at embryonic day (E)10.5 by in situ hybridization (not shown) and qRT-PCR (Fig. 1d). Moreover, there was no compensatory upregulation of Fgf8 in F4;9,17-TKO limb buds at E10.5 (Fig. 1d). These data demonstrate that Fgf8 is su...

pancreatic cancer ͉ paracrine ͉ tumor stroma ͉ SmoM2 ͉ Kras P ancreatic ductal adenocarcinoma (PDA) is one of the most aggressive forms of cancer in the world, with a 5-year survival rate of less than 5%. Potential precursors of PDA include exocrine neoplastic changes, such as pancreatic intraepithelial neoplasms (PanINs), which demonstrate more severe epithelial atypia as they progress toward malignancy. Genetic analyses have linked mutations in human KRAS to PDA (1), and the functional role of oncogenic KRAS in both PDA initiation and progression were subsequently confirmed using genetically engineered animal models of pancreatic cancer (2-9).

SUMMARYIn many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.