How the Hedgehog Outfoxed the Crab: Interference with HEDGEHOG-GLI Signaling as Anti-Cancer Therapy?

Introduction

The enormous success of the last two decades in molecular embryology and developmental genetics have afforded fresh views on old problems. For instance, from a developmental point of view there is an alternative way to think about solid cancers that contrast with the single cell focus derived from molecular and cell biology and their emphasis on the cell cycle and the concepts of transformation and oncogene. One can consider cancers as diseases of patterning affecting stem cell lineages or properties. Within this framework, the funnel hypothesis¹ suggests that the multiple ways to induce a given type of tumor in the mouse and the many functional mutations identified in a given tumor in humans lead to the activation of a single or few events in the cell that affect its position and identity. A one to one relationship between phenotype and final molecular mechanism with some redundancy is proposed for cancer, much as in embryonic development. Moreover, the ability of pathologists to recognize consistent morphologies in tumors and to give appropriate diagnoses is explained by the action of consistent paradevelopmental programs.¹ Concepts like cell competence, specification, niche, developmental history and positional information thus become central to understand, and hopefully treat, cancer. Indeed, under this light, understanding cancer becomes necessary in order to have a complete view of developmental potential.

We can then consider—with caution as we presently lack the perspective that elapsed time affords—what has been learned and achieved on Hedgehog signaling in cancer in recent years. The Hedgehog story is an unfolding one, one that does not yet have a known end, and is only one of several stories,² but so far it is the most promising one.

This story starts with the discovery of the Hedgehog (Hh) gene in Drosophila melanogaster ˜25 years ago, well before Hh signaling was implicated in cancer. The identification of the Hh gene through its mutation, which causes aberrant embryonic patterning giving the mutant larva a ‘hairy’ aspect, and thus named hedgehog,³ was published in 1980. The cloning of the single Drosophila Hh gene was published by several groups in the early '90s,^4-6 with the cloning and identification of Hh genes in vertebrates (Sonic hedgehog (Shh), Indian Hh and Desert Hh in mice and humans) following shortly afterwards.^7-10 The definition of the expression patterns of Hh genes in the developing embryo showed that they are often strikingly restricted to cells and areas previously known to have special patterning activities. The exquisite spatio-temporal regulation of Hh genes in the developing embryo and adult tissues, together with the ability of Hh proteins to elicit defined cell fate changes in a concentration-dependent manner highlighted a revolution in molecular embryology, providing mechanistic explanations for critical events in the formation of the animal body plan. This includes for instance, the patterning and growth of limb buds and of the brain and spinal cord.^7-13

The activity of the Hh pathway involves several steps (Fig. 1).^12-14 Secreted Hh glycoproteins act via the transmembrane proteins Patched1 (Ptch1) and Smoothened (Smo). It is thought that Hh binding to Ptch1 inhibits the repression of Smo by Ptch1 so that Smo functions only when Hh is present. Smo in turn transduces the Hh signal intracellularly to activate the zinc-finger Gli transcription factors.¹³ The Gli proteins are required and sufficient to mediate Hh activity. A number of modulators, positive or negative, some involving negative feedback such Hip1, Gas1 and Ptch1 itself, also regulate Gli activity so that this is tightly controlled in time and space. Because the Hh-Gli pathway is to a large extent linear, activity can be achieved by loss of function of negative factors, such as Ptch1 or the gain of function of positive factors, such as mutations in Smo that may render it insensitive to Ptch1 repression.

Figure 1

Scheme of the HEDGEHOG-GLI pathway. The action of HH, PTCH1 and SMOH at the membrane level is emphasized, together with the site of action of natural and artificial inhibitors. The former include HIP, GAS and PTCH1 itself, while the latter include cyclopamine (more...)

The initial link of HH signaling and familial human cancer was published in the mid-1990s^15,16 linking mutations in PATCHED1 (PTCH1, an inhibitor of HH signaling; Fig. 1), with the rare Basal Cell Nevus syndrome or Gorlin's syndrome.¹⁷ Affected individuals can develop numerous basal cell carcinomas (BCCs) of the skin at an early age as well as medulloblastomas and other tumors, and display additional malformations including odontogenic keratocysts.¹⁷ These studies converged on PTCH1 from the study of its function in flies and mammals¹⁶ and from skin carcinogenesis.¹⁵

The first demonstration of a functional link of HH-GLI signaling with sporadic human cancer was published in 1997, proposing that all human BCCs—the most common type of cancer—derive from the activation of the HH-GLI pathway as all examined BCCs expressed GLI1¹⁸ (Fig. 1), since its transcription is the best marker of a cell's response to HH signaling.¹⁹ In addition to GLI1, GLI2 and PTCH1 (another HH regulated gene²⁰) have also been found to be expressed consistently in sporadic BCCs.^18,21-25

GLI1 was discovered as an amplified gene in a human glioma line in 1987.²⁶ This was followed by the discovery of a related gene in vertebrates (GLI3²⁷) and independently of a single related gene, Cubitus interruptus (Ci), in Drosophila.^28,29 A role for GLI1 in cancer was negated by subsequent findings^30,31 and research on GLI1 waned for several years. However, GLI3 was shortly afterwards identified as the gene mutated in Greig's Cephalopolydactily syndrome^32-34 and later implicated in Pallister-Hall syndrome.^35,36 Patients affected with these diseases display a wide range of phenotypes, including additional digits, skull defects, imperforate anus and hypothalamic growths (hamartomas). The misregulation of post-transcriptionally cleaved C-terminally-deleted forms of GLI3 acting as dominant repressors,^37-41 as first shown for Ci,⁴² has been implicated in these syndromic phenotypes with debated genotype-phenotype correlations.^43,44,228 Aberrant GLI function has also been implicated in other syndromic phenotypes, including VACTERL,^45,46 a syndrome that includes vertebral anomalies, anal atresia, cardiac defects, tracheoesophageal fistula, and renal and limb defects.

Three Gli genes were then cloned from different species, including mice,^47-49 chicks,⁵⁰ frogs,^19,51 and zebrafish.^52,53 They were originally shown to affect neural patterning by mimicking Shh.^19,54 In addition, microinjection of human or frog Gli1 lead to sporadic epidermal and CNS tumors or hyperplasias (Fig. 2).^18,55 Interpretation of GLI1-induced epidermal hyperplasias as tumors related to human BCCs awaited the papers on the involvement of loss of PTCH1 in Gorlin's syndrome^15,16 in which patients develop numerous BCCs.¹⁷

Figure 2

Induction of tumors by misexpressed GLI1. Misexpression of human GLI1 in the developing frog embryo leads to CNS (A,C,D) and skin (B) tumors. Coinjection of GLI1 RNA plus LacZ RNA allows the identification of tumor-forming cells as descendants of the (more...)

Following the finding of familial mutations in PTCH1 in Gorlin's syndrome patients^15,16 and simultaneous with the involvement of HH-GLI signaling in sporadic cancer,¹⁸ mutations in the HH-GLI pathway components PTCH1 and SMOH (Fig. 1) were sought and found in a small fraction of sporadic skin, brain and other tumors,^21,56-67 some of which have been proven to have functional relevance.⁶² There are also reported mutations in Suppressor of fused⁶⁸ (SUFUH; but see ref. 69), a negative modulator of GLI function.^70-72 Mutations in the GLI genes have not been found in tumors, with the exception of beta-actin-GLI1 fusions in a subset of pericytomas.⁷³ These findings provide possible bases for the activation of the HH pathway in a fraction of tumors although many others might derive from epigenetic events.

Various animal models demonstrating the sufficiency of an active Hh-Gli pathway to induce tumor formation (Table 1, Fig. 2) began to be published in 1997 for skin (Shh;⁷⁴ Gli1;^18,75,76 Smo;^62,77 Ptch1;⁷⁸ Gli2^77,79-81), brain (Ptch1;^82,83 Gli1,⁵⁵ Shh+c-Myc;²²¹ Shh+IGF2;²²² Shh+Akt²²²) and muscle (Ptch1⁸³). At the same time, a rational context for the various human tumors now associated with HH-GLI signaling began to be uncovered with the realization that elements of this pathway are expressed in the corresponding normal tissues and that HH-GLI signaling controls growth, patterning and/or homeostasis of developing and adult organs such as the skin^18,74,84,85 and the cerebellum^86-88 (Fig. 3). Indeed, the control of cell proliferation in the late embryonic and perinatal dorsal brain by SHH-GLI signaling may account for its involvement in many types of CNS tumors.⁵⁵

Table 1

Human cancers dependent on sustained HH-GLI signaling.

Figure 3

SONIC HEDGEHOG signaling controls cerebellar patterning and growth and its deregulation leads to medulloblastoma. Schematic representation of a section through the developing cerebellar cortex showing the layered disposition of cell types and the action (more...)

Shh-Gli signaling is not only required for the normal expansion of many types of progenitors but it is also required for the control of neural stem cell behavior in the perinatal and adult rodent brain, including the embryonic neocortex⁸⁹ (Fig. 4), the perinatal and adult hippocampus^90,91 and the subventricular zone of the lateral ventricle of the forebrain^91,92 (Fig. 4). Shh also regulates the proliferation of embryonic spinal cord precursors.⁹³ Similarly, there is evidence that Hh signaling regulates precursor/stem cell lineages and morphogenesis in other tissues and organs, such as the intestine^94-96 and perhaps the skin.^81,97 Together, these findings have raised the possibility that tumors derive from HH-sensitive stem cell lineages, possibly acting at the level of stem cells in some cases or at the level of derived early precursors.

Figure 4

Hedgehog-Gli signaling controls stem cell behavior. A,B) Effects of loss of Shh or Gli2 function in neurosphere cultures derived from mutant mice. E18.5 neocortical neuropsheres produced from Shh or Gli2 null mice show a severe defect in the capacity (more...)

Generally, the best evidence that stem cells exist in tumors come from the transfer of leukemia from a sick mouse to a healthy recipient by the implantation of a few rare cancer stem cells^98-100 and the impressive findings that teratocarcinoma cells can contribute to the development of an entire fertile mouse when transplanted into the inner cell mass of a recipient embryo.^101-103 Here, cancer would appear to be induced by genetic/epigenetic changes that are reversed in the appropriate context or niche. These original experiments demonstrate the stemness of teratocarcinoma cancer cells and their totipotency.

For adult solid tumors, evidence in favor of the existence of cancer stem-like cells derives from the finding that cells with self-renewal properties have been reported in tumor cells from brain and breast.^104-110 Since a small number of glioma cells show clonogenic properties that are regulated by HH signaling (P. Sanchez, V. Clément and ARA, pers. com.) stem cell lineages (stem cells and/or their early derived more restricted progeny) may thus be the target of carcinogens, mutations and epigenetic changes that lead to the initiation of tumorigenesis in the brain and other HH-responsive organs and tissues, such as the epithelial compartment of the prostate, lung or gastrointestinal tract.

The first demonstration that human sporadic cancers require sustained HH-GLI signaling for continued growth derived from the ability to inhibit the proliferation of medulloblastoma cells by blocking HH signaling.⁵⁵ Since then, a rapidly increasing number of sporadic human tumors has been found to express GLI1 and sometimes SHH (Fig. 5), and to be dependent on sustained HH-GLI pathway function (Table 2). These include basal cell carcinomas;^18,111,224 medulloblastomas,^55,112 gliomas⁵⁵ (P. Sanchez, V. Clément and ARA, pers. com), pancreatic tumors,^113,114 small cell lung cancer,¹¹⁵ stomach tumors¹¹⁶ and prostate tumors.^117-120 Additional studies show that a variety of (benign and malignant) tumor cells express HH-GLI pathway components, that tumors are correlated with mutation in genes that affect the pathway and/or that tumor cell lines are sensitive to inhibition of HH signaling. These include breast tumors,¹²¹ sarcomas,¹²² ameloblastomas,¹²³ a subset of pericytomas,⁷³ astrocytomas,^55,124 oral squamous cell carcinomas,¹²⁵ bone exostoses,¹²⁶ neurofibromas,¹²⁷ bladder cancer,¹²⁸ colorectal tumors^129-132 and plasmacytomas²²⁰ among others. The number of tumors and benign tumor-like growths, such as odontogenic keratocycts,¹³³ in which HH-GLI signaling may play a critical role is presently growing at an impressive rate. For example, tumors of liver, colon, rectum, lung and stomach are reported to have low levels of Hedgehog-interacting protein¹³⁴ (HIP1), a HH signaling antagonist.¹³⁵

Figure 5

Consistent expression of SHH-GLI pathway components in sporadic human tumors: basal cell carcinoma, medulloblastoma and prostate carcinoma. Many sporadic human tumors display a constitutively active SHH-GLI1 pathway, as seen by GLI1 mRNA expression, the (more...)

Table 2

Vertebrate animal models of HH-GLI dependent cancers.

The rationale for the initial testing for the requirement of sustained HH-GLI function for tumor maintenance first derived from the timing of appearance of somatic GLI1-induced tumors in tadpoles^18,55 and from the finding that the activity of mutations in SMOH and PTCH1 is inhibited by cyclopamine,¹³⁶ although the latter cannot distinguish between an involvement in tumor initiation or tumor maintenance. The appearance of GLI1-induced tadpole tumors (or hyperplasias) occurred well after all the injected material was degraded,⁵⁵ suggesting the presence of ‘memory’. As the endogenous Gli1 gene was found to be expressed in the induced tumors, it was hypothesized that the activation of the endogenous pathway leading to Gli1 expression was involved in tumor development and could account for the delay in tumor appearance. To test this idea, a morpholino-modified antisense oligonucleotide specific for the endogenous Gli1 mRNA (selectively inhibiting its translation) was coinjected with synthetic human GLI1 RNA. Coinjected embryos developed normally without tumors, demonstrating the requirement of the endogenous Gli1 protein for tumor development.⁵⁵ This result prompted us to originally test the requirement of sustained signaling for tumor maintenance in humans, even if Gli1 was reported to be redundant in mice.^137,138

The requirement of sustained signaling for tumor maintenance and growth has been also recently demonstrated in mice. Conditional expression of Gli2 in basal skin cells leads to the formation of BCCs, which regress when the expression is turned off after tumor formation.⁸¹ This result is in line with previous data on the dependence of mouse tumors on the maintained expression of the inducing oncogene.^139-141

Inhibition of HH-GLI function in human tumor cells was first achieved with cyclopamine,⁵⁵ is a plant alkaloid with an overall resemblance to cholesterol that is produced by Veratrum californicum, a corn lily.^142-145 It was named cyclopamine because it is the active compound, along with jervine, responsible for the production of cyclopic embryos and newborns after ingestion of V. californicum by pregnant range and farm animals.^144,146,147 Cyclopamine-induced cyclopia phenocopies the loss of Shh in mice¹⁴⁸ and humans^149,150 and it selectively inhibits the HH-GLI pathway,¹⁵² binding and blocking the function of SMOH (Fig. 1).¹⁵³

Inhibition of HH-GLI signaling by noncyclopamine small molecule agents^154,155 that, like cyclopamine, target SMOH has been achieved in mouse basal cell carcinoma punch explants and cell lines.^111,155 Antibodies against SHH¹⁵⁶ and immunization with HIP1 have also been used.^116,117,157 At the other end of the signaling pathway, inhibition has been achieved by targeting Gli1 with antisense oligonucleotides in skin and CNS tumors in tadpoles⁵⁵ and with siRNAs in human prostate cancer¹¹⁷ (Fig. 6) and in brain tumor cells (P. Sanchez, V. Clément and ARA, pers. com). Targeting GLI1 offers that best therapeutic outlook as this would inhibit activation of the pathway by any event acting at any level. For instance, alterations in SUFUH have been observed in different tumors^68,119 and tumors arising from such events would be insensitive to inhibitors acting on SMOH, as SUFUH acts downstream of SMOH. Indeed, tumor cells insensitive to cyclopamine but dependent on GLI1, as assessed by RNA interference (RNAi), have been found.¹¹⁷

Figure 6

Inhibition of human prostate cancer cell proliferation by cyclopamine and GLI1 RNA interference. Cyclopamine inhibits the proliferation of primary in situ prostate tumors (A) and of metastatic prostate cancer cell lines (C), as measured by BrdU incorporation, (more...)

Inhibition of cancer growth through interference with HH-GLI signaling has been shown for established cell lines and for primary cultures from the operating room^{55,112,113,115-118} (Table 2) as well as for mouse grafts.^{112,113,115,116,118} It is important to note, however, that while orthografts are valuable, the predictive value of subcutaneous xenografts and allografts is questionable at best and equal or inferior to primary cell cultures.^158,159

Genetic mouse models of endogenous tumors, however, provide excellent tools for preclinical assessments in a mammal. In this sense, the efficient development of medulloblastomas in all Ptch1+/-;p53-/- mice¹⁶⁰ provides a valuable model with a genetically engineered endogenous tumor. Using this model it has been recently shown that systemic interference of HH signaling in Ptch1+/-;p53-/- adult mice leads to tumor inhibition and regression using a synthetic small molecule antagonist¹⁶¹ of SMOH function or using the natural alkaloid cyclopamine¹⁶² (Fig. 7). Similarly, oral cyclopamine treatment was preventive for uv-induced BCC development in Ptch1+/- mice.²²⁴ Together, these findings set the stage for the initiation of clinical trials on human patients suffering from incurable terminal cancers such as metastatic prostatic and pancreatic tumors or glioblastoma multiforme.

Figure 7

Beneficial anti-cancer effects of systemic in vivo cyclopamine treatment in mice. Ptch1+/-;p53-/- mice develop medulloblastoma and die at 2 months of age. Treatment with cyclopamine through systemic intraperitoneal injections for 30 days starting at 1 (more...)

Inhibition of HH-GLI function appears to be effective for all tumor grades of all ages and subgroups of a given type that is HH-GLI signaling dependent. For example, primary culture of in situ prostate tumors of different grades and cells lines derived from distant metastatic lesions¹¹⁷ (Figs. 5, 6), as well as primary culture of fresh prostatic metastatic lesions from postmortem specimens,¹¹⁸ are sensitive to cyclopamine. These and similar findings in other tumor types is amazing and suggest that the HH-GLI pathway is an essential basis on which a tumor is built and that no tumor cell can exist without sustained signaling, be it autocrine or paracrine, leading to positive GLI function (Fig. 8). HH-GLI signaling may thus be a critical sensor of the ‘fitness’ of a cell, with tumor cells requiring a high fitness.

Figure 8

Schematic representation of the proposed causal relationship of the abnormal state of HH-GLI signaling in prostate cancer. Homeostatic HH-GLI signaling participates in the normal maintenance and regeneration of the adult prostatic epithelium. Enhanced (more...)

Metastatic lesions may derive from the acquisition of cell-autonomous pathway activation allowing efficient activity independent of the original niche¹¹⁷ and/or from increased overall signaling levels (Fig. 8).¹¹⁸ High levels of GLI1 function lead to high metastatic behavior in xenografts.¹¹⁸ Intrinsic/elevated signaling may then affect genes involved in epithelial-mesenchymal transitions, such as C-Myc, N-Myc¹⁶³ and Snail,¹⁶⁴ genes which are responsive to Hh-Gli activity^118,165-167 to start the metastatic voyage and departure from the original tumorigenic niche. c-Myc also cooperates with Shh signaling in tumorigenesis.²²¹ The context of the site of origin of the tumor might also determine the site of metastatic growth, perhaps not just in a random selective manner but also in a directed way, possibly recapitulating aspects of normal cell migration, such as that of the neural crest during embryogenesis.

Unexpectedly for its protocols and expediency, two publications report beneficial effects of cyclopamine on BCCs and psoriasis in human patients.^168,169 If confirmed and extended, these results are in perfect agreement with the expected outcome of interference with HH signaling as a novel therapeutic approach in humans. Toxicology and side effect issues need to be addressed before general and systemic treatments can be started but cyclopamine could indeed be a perfect leading compound.

Systemic cyclopamine treatment could cause a number of side effects that might include problems with the gastric mucosae, hair growth or renewal and production of new neurons. However, these are not worse than the side effects of present-day chemotherapy and local, topical cyclopamine treatment did not affect hair follicles adversely.¹⁶⁸ Moreover, a minimal treatment period required to induce growth arrest and death of tumor cells may result in transient secondary effects that can be compensated following cessation of the treatment: mice treated for several weeks with cyclopamine^162,224 or a different SMOH antagonist¹⁶¹ appear normal.

In principle, however, the best therapies could derive from inhibition of GLI function (Fig. 1), which as it is the last element of the pathway would appear to be the best target. Moreover, GLI proteins are sufficient for tumor induction in animal models^18,55,75,79 and might respond to additional tumorigenic inputs other than HH.¹⁷⁰ Given that the GLI code^13,171,172 is critical for cell fate and cancer, and that it is modulated by several kinases (Fused, PKA, GSK3β, CK1 and GRK2)^173-183 and many cofactors, inhibitors of GLI function may target the GLI proteins themselves or such modifying proteins (Fig. 1). These include DYRK1,¹⁸⁴ intraflagellar transport proteins,¹⁸⁵ ZIC proteins,^186,187 REN,¹⁸⁸ DZIP1 (Iguana),^189,190 RAB23,¹⁹¹ FKBP8¹⁹² and SUFUH70-^72,193,194 and BEGH.²²³ Approaches with antagonist (for positive factors) or agonist (for negative regulators) small molecules as discussed above, peptides mimicking dominant-repressive C-terminus deleted GLI proteins^37,38 and RNA interference¹¹⁷ (Fig. 7) will very likely lead to the production of efficient, specific and potent inhibitors for patient therapy. Additional beneficial strategies could derive from finding the key targets regulated by HH-GLI signaling the inhibition of which may partially recapitulate loss of activating GLI function. Moreover, large-scale gene profiling, microchip-based diagnoses¹⁹⁵ and individual treatment regiments could also lead to more focused and thus better outcomes.

Given the results summarized here, it is difficult to temper enthusiasm for the development of a rational wide-spectrum tumor therapy derived from research on molecular embryology, plant-induced teratology, cancer biology and developmental genetics. Such a therapy would target cancers from brain, lung, pancreas, prostate, muscle, stomach, skin and other organs and of any grade, including most importantly metastatic cancer.

This story is still ongoing and a conclusion has not been reached. At present, pressing questions include the following. How many different types of human tumors depend on HH-GLI signaling? Do tumors derive from stem cells and/or from de-differentiating cells that acquire stem cell properties? What are the paradevelopmental programs that dictate tumor initiation, growth and metastasis? Do HH proteins act as mitogens and/or morphogens in the control of stem cell behavior? Do they contribute to the definition of a normal and tumorigenic stem cell niche? Is there a specific GLI code for each tumor type or a general one that is modified in a context-dependent manner by local cofactors? Is there a correlation of levels of GLI expression/function with tumor grade or character? What are the targets that execute the programs instructed by GLI proteins? Can specific and effective inhibitors of HH-GLI function be produced for patient treatment? Will these have limited and tolerable side effects? Will HH-GLI inhibitors also affect and kill cancer stem cells, thought to be responsible for recurrence? Can cancer result from repeated injury and the eventual perversion of the behavior of stem cells recruited for repair? Can one develop not only anti-cancer but also preventive therapies?

Other questions for which we begin to have some insights include: Do other oncogenic inputs affect or regulate HH-GLI function or vice versa? What are the mechanisms that guide cancer-causing events towards regeneration in some instances? We know that there are several intercellular communication systems that have been implicated in cancer, including FGF, WNT, NOTCH and TGFβ signaling.² How these interact with HH-GLI function is not yet clear although we know, for instance, that Gli proteins regulate batteries of Wnt genes,¹⁹⁶ that FGF signaling can regulate Gli2 and Gli3¹⁷⁰ and that Gli2 can be responsive to Notch signaling.^197,198 And as far as cancer and regeneration is concerned, insights derive from a variety of sources. For example, on the one hand, treatment of a newt with highly tumorigenic compounds after lens extirpation leads to normal regeneration of the lens from the dorsal iris, but tumor formation from the ventral, nonregenerative ventral iris.¹⁹⁹ And on the other hand, some mammalian tumors can be induced to differentiate. For instance, Shh- and Gli2-induced BCCs can differentiate into hair follicle components if placed adjacent to a normal environment,^74,81 and teratocarcinomas participate in the formation of a normal mouse when transplanted into a blastocyst.^101-103 The developmental history, competence and position of a cell could thus turn tumorigenic into (re)generative information (and vice versa!).

More specifically, these issues on signaling, pattern formation and cancer have lead to renewed interest in homeobox genes. The demonstration of the control of Xhox3 (Evx1) expression by Gli2 and Gli3,¹⁷⁰ the regulation of HoxD genes by Gli3,^200,201 a physical and functional interaction between HoxD12 and Gli3²⁰² and the genetic interactions between Proboscipedia (HoxA2/B2) and Ci,²⁰³ the Gli homologue in Drosophila, suggests the possibility that cell number may be organized to lead to pattern through an interaction between Gli and homeobox proteins. The latter being often regulated by extracellular inputs and best known as organizers of body pattern.^204,205 This could explain several unresolved observations, for example, the role of HoxB1 in the control of Shh-mediated D-V pattern in rhombomere 1,²⁰⁶ the ability of HoxB4 to induce hematopoietic stem cell proliferation,^207,208 and the involvement of Msx1 in Shh signaling during tooth development.²⁰⁹ The roles of HH-GLI signaling in cancer and stem cell/precursor proliferation (see above), together with the documented participation of homeobox proteins in cancer,^210,211 raise the possibilities that these factors regulate size and shape in embryos and homeostasis and regeneration in adults, and that their regulation underlies a unified principle in cancer and development. This hypothesis could also explain evolutionary constrains in body plan and cancer selection.²¹² Patterning targets/cofactors, such as HOX proteins, in addition to the GLI proteins and their modulators, could thus also be interesting targets of anti-cancer, preventive and pro-regenerative therapies.

In contrast with the inappropriate activation and/or maintenance HH-GLI signaling in cancer, its ability to regulate neurogenesis and brain stem cell behavior,^{89-92,186,213} raises new and exciting prospects to increase cell number by acting positively on this signaling pathway. This could be critical to increase the pool of cells that is undergoing degeneration, and thus reverse cell loss as in Parkinson's disease,²¹⁴ or to provide exogenous stem cells to increase repair. Indeed, the ability of SHH to act as a motor neuron survival factor²¹⁵ and of SHH and GLI1 to act as protective factors for dopaminergic neurons^216,217 suggest novel therapies, which may also include the manipulation of homeobox proteins.^226,227 Given the adult expression of components of the HH-GLI signaling pathway in the adult brain it is not inconceivable that its deregulation could be involved in additional mental diseases and disorders. Clearly, much awaits to be discovered although the data gathered up to this point already strongly suggest that manipulation of the SHH-GLI1 pathway will lead to cancer treatment, rationally reducing cell number, and to the treatment of degenerative diseases, rationally increasing cell number. Current results are very encouraging and enthusiasm is high. With caution, the goal of developing rational therapies based on knowledge derived from research on flies, lilies, tadpoles, mice, stem cells and human disease is within reach.

Acknowledgements

I am very grateful to Nadia Dahmane, Barbara Stecca, Pilar Sánchez, Verónica Palma, José Mullor, Virginie Clement, Christophe Mas and Ivan Radovanovic for discussion and/or comments on the manuscript, and to all present and previous lab members for sustained intellectual excitement. Work from the author's laboratory was supported by grants from the NIH, Pew Scholars Program, Concern Foundation, Jeantet Foundation, March of Dimes and a Hirschl Award. The reader is encouraged to perform PubMed searches to complement the cited literature and establish time lines as not all references could be included.

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