Interactive Fly, Drosophila

Hedgehog, cell proliferation and basal cell carcinoma

Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1 mutant mice obscures the roles of ptc1 in later stages of development. ptc1 homozygous mutant embryos were partially rescued using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allows survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gives rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly is preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud (Milenkovic, 1999).

Transgenic mice that overexpress ptc1 are consistently smaller than control mice and the opposite is true as well -- larger body size is associated with heterozygosity at the ptc1 locus, just as it can be in humans. Another tumor suppressor gene has similar effects: overexpression of the retinoblastoma gene in mice induces dose-dependent growth retardation, as early as E15. The size of an animal generally reflects cell numbers and therefore a balance between cell proliferation and cell death. It is believed that substantially fewer cells are formed during the development of MT-ptc1 mice. Shh has mitogenic effects in several tissues including presomitic mesoderm, retina and cerebellum. Another Hh protein, Ihh controls growth in the cartilage, and Ihh-deficient mice exhibit short-limbed dwarfism. Overexpression of Hip1, another negative regulator of Hh signaling, in the cartilage, leads to a shortened skeleton that resembles that seen in Ihh loss-of-function mutants. ptc1 may counteract mitogenic activities of one or all of the Hh proteins to restrict body size. The embryos are remarkably well proportioned, so either Ptc exerts growth control in many locations or all tissues are somehow coordinated in size with those directly affected by ptc1. The generality of the effect of Ptc1 on body size raises the possibility of growth control unrelated to the Hedgehog genes. For example, there may be Ptc1 actions that cannot be blocked, or are never blocked, by a Hh signal. Since the MT-ptc1 transgenic embryos are different in size as early as the eleventh day of embryogenesis, the effects of Ptc1 are likely to precede, and be independent of, growth hormone. Major determinants of mouse embryonic growth are insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), and their cognate type 1 receptor (IGF1R). Mutations in all three genes have dwarf phenotypes. For the IGF-II and IGF-1R mutations, significant reduction in embryo size first starts at about E10.5. The IGF-I phenotype does not become evident until E13.5. Since growth-modifying effects of IGFs are apparent as early as E10.5 of embryogenesis, the IGF signaling circuitry may be involved in growth retardation effects of ptc1 (Milenkovic, 1999).

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 Drosophila suggest that overproduction of Sonic hedgehog (SHH), the ligand for PTC, will mimic loss of ptc function. 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 (Oro, 1997).

Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC) is implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. Retroviral transduction of normal human keratinocytes was used to constitutively express SHH. SHH-expressing cells demonstrate increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia (Fan, 1997).

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. Expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, it has been found that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis (Reddy, 2001).

SHH is not required for the positioning of follicles, but plays essential roles in the regulation of follicular proliferation and formation of the dermal papilla. Previous data have indicated that expression of Shh in hair follicles is regulated by canonical WNT signaling and WNTs 10a and 10b are the most likely candidates for WNTs that control Shh expression in hair follicle morphogenesis. However, Wnt genes are also targets of SHH in several developmental systems and consistent with this observation, it has been found that expression of Wnt5a in developing hair follicles requires SHH. This result suggests that WNT5a may mediate some of the effects of SHH in hair follicle morphogenesis, a hypothesis supported by the fact that both WNT5a and SHH are capable of regulating proliferation. Since later stages of hair follicle morphogenesis are abnormal in Shh minus mutants, the question of whether expression of Wnt5a in mature hair follicles is also regulated by SHH could not be addressed; however, like Wnt5a, Shh is expressed in inner root sheath cells in anagen follicles (Reddy, 2001).

The finding that Wnt5a is a target of SHH signaling in hair follicles has important implications for the study of basal cell carcinoma (BCC), a human skin tumor that occurs with high frequency in Caucasian populations. BCC results from inappropriate activation of the SHH pathway in epidermal cells and is frequently associated with mutations in the gene encoding the SHH receptor PTC1. Like developing hair follicles, BCCs show elevated expression of PTC1 and GLI1, which encodes a transcriptional effector of SHH signaling. In addition to activation of the SHH signaling pathway, BCCs share many common characteristics with immature hair follicles, including similar histology, ultrastructure and patterns of keratin gene expression, suggesting that SHH activates the same downstream target genes in BCCs and hair follicles. BCC can be mimicked in transgenic mice by over-expression of Shh, Gli1 or Gli2 in the epidermis and Wnt gene expression is directly regulated by SHH via GLI transcription factors in Drosophila and zebrafish embryos. However, Wnt targets of the SHH pathway in BCC have not been identified. Given the similarity of BCC to immature hair follicles, the results presented here predict that Wnt5a is upregulated in BCC. Nuclear localization of ß-catenin is not observed in BCC consistent with classification of WNT5a as a Class II WNT (Reddy, 2001).

The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. This study describes an unexpected role of the HMG-box-containing gene Sox9 in hair development. Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, it was demonstrated that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, this study shows that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrate overexpression in skin tumors in mouse and man. It is concluded that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma (Vidal, 2005).

Temporally and spatially constrained Hedgehog (Hh) signaling regulates cyclic growth of hair follicle epithelium while constitutive Hh signaling drives the development of basal cell carcinomas (BCCs), the most common cancers in humans. Using mice engineered to conditionally express the Hh effector Gli2, it was shown that continued Hh signaling is required for growth of established BCCs. Transgene inactivation leads to BCC regression accompanied by reduced tumor cell proliferation and increased apoptosis, leaving behind a small subset of nonproliferative cells that could form tumors upon transgene reactivation. Nearly all BCCs arise from hair follicles, which harbor cutaneous epithelial stem cells, and reconstitution of regressing tumor cells with an inductive mesenchyme leads to multilineage differentiation and hair follicle formation. These data reveal that continued Hh signaling is required for proliferation and survival of established BCCs, provide compelling support for the concept that these tumors represent an aberrant form of follicle organogenesis, and uncover potential limitations to treating BCCs using Hh pathway inhibitors (Hutchin, 2005).

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells. Human gliomas display a stemness signature and demonstrate that Hedgehog (Hh)-Gli signaling regulates the expression of stemness genes in and the self-renewal of CD133+ glioma cancer stem cells. Hh-Gli signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of Hh-Gli signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. These results reveal the essential role of Hh-Gli signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities (Clement, 2007).

Pancreatic ductal adenocarcinoma (PDA) constitutes a lethal disease that affects >30,000 people annually in the United States. Deregulation of Hedgehog signaling has been implicated in the pathogenesis of PDA. To gain insights into the role of the pathway during the distinct stages of pancreatic carcinogenesis, a mouse model was established in which Hedgehog signaling is activated specifically in the pancreatic epithelium. Transgenic mice survived to adulthood and developed undifferentiated carcinoma, indicating that epithelium-specific Hedgehog signaling is sufficient to drive pancreatic neoplasia but does not recapitulate human pancreatic carcinogenesis. In contrast, simultaneous activation of Ras and Hedgehog signaling caused extensive formation of pancreatic intraepithelial neoplasias, the earliest stages of human PDA tumorigenesis, and accelerated lethality. These results indicate the cooperation of Hedgehog and Ras signaling during the earliest stages of PDA formation. They also mark Hedgehog pathway components as relevant therapeutic targets for both early and advanced stages of pancreatic ductal neoplasia (Pasca di Magliano, 2007).

Sonic hedgehog and neural stem cells

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, conditional null alleles of both the Sonic hedgehog and Smoothened genes were examined. While the removal of Shh signaling in these animals results in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus is dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals form significantly fewer neurospheres. The loss of hedgehog signaling also results in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon (Machold, 2003).

In order to explore which populations in the postnatal (P15) brain might be affected by the loss of hedgehog signaling, the distribution of Shh and its transcriptional targets Ptch1 and Gli1 was examined. Immunostaining using a Shh-specific antibody revealed that Shh is broadly distributed in the P15 telencephalon. The most prominent areas of Shh immunoreactivity are in the ventral telencephalon where embryonic Shh expression is observed. Specifically, Shh-positive cells are seen within the ventral accumbens, entorhinal cortex, and ventral septum, along the path transited by cells in the rostral migratory stream. Furthermore, within layer 3 of the cortex and along the corpus callosum Shh immunoreactivity is observed. Finally, a population of axons in the medial septum is also immunopositive for Shh. This observation is consistent with the observation that the septal (i.e., fimbria) fibers projecting to the hippocampus are a source of Shh within this structure. Shh staining is seen in the white matter tracks in the CA3 and hilus regions of the hippocampus. Mice heterozygous for a LacZ reporter under the control of the endogenous Shh promoter display a similar pattern of cellular Shh expression. In addition, these mice revealed the existence of Shh-expressing cells within the hilus of the dentate gyrus, adjacent to the hippocampal stem cell population. Since this staining was not evident in antibody visualization of Shh, it seems likely that these cells only express low levels of Shh and require the enzymatic amplification provided by LacZ staining to visualize. The expression of LacZ directed through either the Ptch1 or Gli1 loci is generally consistent in indicating in which populations the hedgehog pathway is activated. The expression of LacZ directed by the Ptch1 loci is broader than that seen from the Gli1 loci. The observation that Gli1 expression more closely matched sources of endogenous Shh suggests that Gli1 is the more accurate readout of Shh activation. Furthermore, embryonic studies indicate that all Gli1 expression is Shh dependent, whereas Ptch1, the Shh receptor, is expressed at basal levels in the absence of any direct Hedgehog signaling input (Machold, 2003).

The data suggests that Shh signaling has only modest effects on the general growth of ventral telencephalic regions after E12.5. In contrast, it is essential in the dentate gyrus and the olfactory bulb, regions in which neurogenesis continues in the adult. Specifically, Smo^n/c;N^cre mice display a marked decrease in the levels of proliferation in both these areas. In addition, in the SVZ, apoptotic cell death is dramatically increased. Within the hippocampus Shh signaling acts as a mitogen for adult progenitors residing in the dentate gyrus. While the data support the contention that Shh is required for the maintenance of hippocampal and SVZ progenitors, it is less clear that within the SVZ Shh acts directly as a mitogen. Although both the SVZ and dentate gyrus of Smo^n/c;N^cre mutants appear to have fewer progenitors, Shh alone is unable to support neurosphere formation or expansion from either wild-type or Smo^n/c;N^cre SVZ progenitors, whereas Shh appears to be able to substitute for FGF in the expansion of dentate gyrus precursors in rat. Similarly, while almost all hippocampal cells labeled by a Brdu pulse 2 hr prior to sacrifice at P15 expressed Gli1, only a small subpopulation of similarly labeled cells in the SVZ express this direct target of Shh signaling. Nonetheless, in both the SVZ and hippocampus, increasing levels of Shh signaling increase cell proliferation. Hence, it will require further work to determine the precise actions of hedgehog signaling in maintaining telencephalic progenitor regions (Machold, 2003).

Stem cells are crucial for normal development and homeostasis, and their misbehavior may be related to the origin of cancer. Progress in these areas has been difficult because the mechanisms regulating stem cell lineages are not well understood. The role of the SHH-GLI pathway in the developing mouse neocortex has been investigated. The results show that SHH signaling endogenously regulates the number of embryonic and postnatal mouse neocortical cells with stem cell properties, and controls precursor proliferation in a concentration-dependent manner in cooperation with EGF signaling. Shh^-/- mice die at birth showing overt signs of cyclopia and lacking all ventral CNS cell types. Their dorsal-only CNS comprises an Emx1⁺, Tbr1⁺ forebrain cortex. The Shh^-/- cortex produced neurosphere (nsp) cultures in full media, but these were fewer and smaller than those from wild-type cortices, and contained fewer BrdU⁺ cells. Analyses of gene expression confirmed the loss of Shh transcripts in the few Shh^-/- nsps that formed (representing a small pool of viable cells). A decrease in Ptch1 and Dhh expression was detected, whereas the expression of Ihh, Gli1 and Gli2 were unchanged, and the expression of Gli3 expression was slightly higher. These Shh^-/- nsps expressed nestin and were tripotential, as judged by the ability to differentiate as Tuj1⁺ neurons, GFAP⁺ astrocytes or O4⁺ oligodendrocytes. Cloning assays showed that Shh^-/- nsps contain approximately one quarter of the number of nsp-forming stem cells of wild-type nsps at E15.5. At E18.5, there were very few, if any, mutant nsps. Gli2^-/- mice also die at birth, displaying defects in multiple organs (Palma, 2004).

Novel dorsal brain phenotypes of Gli2^-/- mice were found at mid and late gestation stages in an outbred background. Gli2 null embryos present a variably penetrant severe phenotype, displaying excencephaly by E13.5 (also seen at E17-E18.5), and a consistent milder phenotype characterized by expanded but thinner telencephalic vesicles, most clearly seen posteriorly, and an overtly reduced tectum and cerebellum. Focus was placed on the non-exencephalic Gli2^-/- mice. Histological analyses showed that E18.5 Gli2^-/- telencephalic vesicles have a thinner proliferative zone (an ~30-50% reduction of the vz/svz). Gli2^-/- mice have fewer BrdU⁺ precursors in the cortex at mid and late gestation periods, suggesting defects in neuronal as well as glial cell populations. The decrease is most notable in the deeper proliferative area (the svz). Local variations without a clear pattern in the density of BrdU⁺ nuclei were also observed, indicating an additional degree of neocortical disorder in these mutant mice. TUNEL and activated caspase 3 analyses did not show an increase in apoptosis (Palma, 2004).

Gli2^-/- neocortices gave rise to nsps, containing Nestin⁺ cells that were tripotential. However, at late embryonic stages mutant nsps progressively became smaller, more delicate, and showed more blebbing than wild-type nsps. Gli2^-/- nsps decreased in numbers during culture and, after a few passes, they were rare, and all died soon after. The Gli2^-/- nsps surviving at passage ~2-4 lacked Gli1 expression and showed downregulation of Ihh and Dhh expression. Shh expression was unchanged, whereas Gli3 and Ptch1 expression was reduced. The expression of Egfr was also reduced. Cloning assays in the presence of EGF and FGF showed that there was an ~10-fold decrease in the number of Gli2^-/- cells able to form secondary nsps, as compared with wild-type cells. These findings identify a crucial mechanism for the regulation of the number of cells with stem cell properties that is unexpectedly conserved in different stem cell niches (Palma, 2004).

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Shh has been shown to be required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. Two populations of Gli1⁺ Shh signaling responding cells have been identified: GFAP⁺ SVZ stem cells and GFAP^- precursors. Consistently, Shh regulates the self-renewal of neurosphere-forming stem cells and it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, these data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis (Palmo, 2005).

YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation

Medulloblastoma is the most common solid malignancy of childhood, with treatment side effects reducing survivors' quality of life and lethality being associated with tumor recurrence. Activation of the Sonic hedgehog (Shh) signaling pathway is implicated in human medulloblastomas. Cerebellar granule neuron precursors (CGNPs) depend on signaling by the morphogen Shh for expansion during development, and have been suggested as a cell of origin for certain medulloblastomas. Mechanisms contributing to Shh pathway-mediated proliferation and transformation remain poorly understood. This study investigated interactions between Shh signaling and the recently described tumor-suppressive Hippo pathway in the developing brain and medulloblastomas. Up-regulation is reported of the oncogenic transcriptional coactivator yes-associated protein 1 (YAP1; homolog of Drosophila Yorkie), which is negatively regulated by the Hippo pathway, in human medulloblastomas with aberrant Shh signaling. Consistent with conserved mechanisms between brain tumorigenesis and development, Shh induces YAP1 expression in CGNPs. Shh also promotes YAP1 nuclear localization in CGNPs, and YAP1 can drive CGNP proliferation. Furthermore, YAP1 is found in cells of the perivascular niche, where proposed tumor-repopulating cells reside. Post-irradiation, YAP1 was found in newly growing tumor cells. These findings implicate YAP1 as a new Shh effector that may be targeted by medulloblastoma therapies aimed at eliminating medulloblastoma recurrence (Fernandez-L, 2009).

This study demonstrates that YAP1 and its transcriptional partner, TEAD1, are highly expressed in Shh-driven medulloblastomas in both humans and mice. YAP1 is amplified in a subset of human medulloblastomas -- specifically, SHH-associated medulloblastomas. Moreover, YAP1 expression is up-regulated by the Shh pathway in proliferating CGNPs. Shh signaling regulates YAP1 nuclear localization through its binding to IRS1, and YAP1 activity promotes CGNP proliferation, at least in part through interactions with TEAD1. In mouse medulloblastomas, YAP1 protein localized to the cells occupying the perivascular niche (PVN) that have been proposed to have cancer stem cell properties. Indeed, YAP1-positive cells remain alive and disseminated through the tumor after the tumor bulk cells have been eradicated by radiation. These findings mark YAP1 as a mediator of normal proliferation in the developing cerebellum, and as a potential target for medulloblastoma therapies aimed at eliminating tumor-reinitiating cells (Fernandez-L, 2009).

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