Christina P. Ahn

A paracrine requirement for hedgehog signalling in cancer

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.

Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma.

A Smooth(ened) Path to Drug Resistance The Hedgehog (Hh) signaling pathway has emerged as a key contributor to the growth of medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that ramps down this signaling pathway by binding to the Hh pathway component Smoothened, was recently shown to induce rapid and dramatic tumor regression in a patient with metastatic medulloblastoma, but the tumor eventually developed resistance to the drug. Yauch et al. (p. 572, published online 3 September) show that resistance arose because the tumor acquired a mutation in Smoothened that disrupts binding of the drug. Identification of this resistance mechanism may facilitate the design of next-generation drugs for this type of cancer. A mutation that prevents binding of a promising drug lead to its target protein confers resistance in a human brain tumor. 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.

Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis

The Hedgehog (Hh) pathway has been implicated in pancreatic cancer but its role remains controversial. To delineate the cell populations able to respond to Hh ligand stimulation, we expressed an oncogenic allele of Smoothened (SmoM2) to cell autonomously activate Hh signaling in the mouse pancreas. Surprisingly, we found that expression of SmoM2 in epithelial cells was not able to activate the pathway and had no impact on pancreatic development or neoplasia. In contrast, activation of Smo in the mesenchyme led to Hh pathway activation, indicating that only the tumor stroma is competent to transduce the Hh signal. Using a Ptc-LacZ reporter mouse, we show that Hh signaling is active in stromal cells surrounding Hh-expressing tumor epithelium in various mouse pancreatic cancer models. Activation of the Hh pathway in the tumor stroma of human pancreatic and metastatic cancer specimens was confirmed by quantitative RT-PCR of microdissected tissue samples. These data support a paracrine model of Hh-mediated tumorigenesis, in which tumor cells secrete Hh ligand to induce tumor-promoting Hh target genes in adjacent stroma.

Genetic evidence that FGFs have an instructive role in limb proximal–distal patterning

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. Fibroblast growth factor (FGF) gene family members are key AER-derived signals, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER. Here we demonstrate that mouse 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; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P–D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P–D patterning can be integrated with the concept of early specification to explain the genetic data presented here.

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In 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.

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation.

Stromal Indian Hedgehog Signaling Is Required for Intestinal Adenoma Formation in Mice

BACKGROUND & AIMS Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of β-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.

Brachydactylic multiple delta phalanges plus syndrome

Delta phalanges are unusual, shortened bones of the hands and feet with abnormal epiphyses and diaphyses. Here, we report on a patient with a unique multiple congenital anomaly syndrome that includes brachydactyly consisting of multiple delta phalanges in several digits of the hands and feet. The patient, who was born to consanguineous parents, had several other congenital anomalies, including butterfly vertebrae, craniofacial dysmorphism, and coarctation of the aorta. We have called this distinctive condition “brachydactylic multiple delta phalanges plus syndrome.” Although no other member of the family had obvious hand or foot anomalies, several siblings had other malformations. Possible modes of inheritance in this family include variable expression of a recessive or de novo dominant condition.

19-P008 SHH is a master regulator of stem cell-driven continuous growth of the mouse incisor

in vertebrates and to determine the molecular and cellular basis for such currents. Methods: We use zebrafish caudal fin as an adult regeneration model. Specific ion flux measurements are done using a non-invasive Ion-Specific Scanning Microprobe coupled with transcriptional profiling and genetic functional analysis. Results: Our data points to an important role for protons (H[+]) during the regeneration process. H[+] effluxes are triggered during wound healing and maintained throughout regeneration. Microarray analysis revealed the proton pump V-ATPase as a putative mediator of such H[+] fluxes and we observed that it is expressed in the blastema. We are now exploring this proton-based mechanism with genetic and pharmacological approaches coupled with advanced pH imaging. Other ions are also under study. Conclusions: Overall, our results reveal that important ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies and drug screenings, both in regenerative and developmental medicine, and in cancer therapy.