hh signaling is essential for development of all vertebrates and drives proliferation , migration , and differentiation of progenitor cells to pattern organ development ( varjosalo and taipale , 2008 ) . despite the critical nature of hh signaling , how hh mediates tumor proliferation
hh pathway activation begins when the hh ligand binds to and inhibits the transmembrane receptor patched1 ( ptch1 ) , allowing the signal transducer smoothened ( smo ) to activate gli transcription factors and amplify hh target gene expression .
so far , all of the nuclear events ascribed to hh occur through the gli transcription factors , with gli1 acting predominantly as an activator , gli3 acting predominantly a repressor , and gli2 possessing both repressive and activator functions . although most of the major components of the hh pathway have been known from three decades of work in drosophila melanogaster , the role of the primary cilium , a microtubule - based organelle , as a vertebrate - specific regulator of the pathway has been appreciated only recently ( huangfu et al . , 2003 ) .
abnormalities within ciliary components lead to a variety of developmental defects and cancer predisposition syndromes called  ciliopathies ,  which include hh - specific phenotypes ( hildebrandt et al .
the primary cilium is necessary to amplify hh signal in vertebrates , in addition to regulating gli processing and activity ( hui and angers , 2011 ) . in the absence of hh ligand , ciliary ptch1 suppresses smo activity and entry into the cilium , allowing the negative regulator suppressor of fused ( sufu ) to inhibit gli activity ( fig .
ptch1 transiently exits the primary cilium after inactivation by hh ligand , allowing entry of active smo into the cilium that binds to and inhibits sufu , resulting in gli transcriptional activity ( fig .
ciliary components actively transduce hh signal , and mutations that disrupt intraflagellar trafficking , such as ift25 , activate the pathway through inappropriate accumulation of smo and ptch1 along the ciliary shaft and loss of gli at the ciliary tip ( keady et al . , 2012 ) .
moreover , primary cilia regulate gli processing and inhibition through sufu , which determines the level of pathway activation ( humke et al .
( b ) hh ligand activates signaling by binding and inhibiting ptch1 , allowing smo to suppress sufu and activate gli transcription factors to turn on hh target genes .
( c ) common pathway - dependent genetic mutations that lead to hh - dependent tumors include ptch1 inactivation , smo activation , or inappropriate shh expression .
( d ) smo antagonists such as vismodegib suppress hh activation to prevent tumor growth .
( e ) genetic escape pathways that evolve during smo antagonist treatment include smo point mutations that prevent smo  drug interaction or ( f ) gli target gene amplification of gli2 or ccnd1 .
( g ) compensatory escape pathways that have evolved include inappropriate activation of s6k1 that prevents sufu inhibition of gli and ( h ) pi3k pathway up - regulation leading to inappropriate gli activity through currently unknown mechanisms .
overwhelming data exists for the dependence of bcc and medulloblastoma growth on hh pathway activation .
for instance , bccs , which are invasive epithelial tumors , originate from activating mutations in the hh pathway in progenitor cells of the interfollicular epidermis and hair follicle .
they retain basal keratinocyte histology , and invade as either branching or nest - like nodular structures .
1 c ) comprise the bulk of bcc cases ( epstein , 2008 ; gomez - ospina et al . , 2012 ) .
induction of hh target genes in human skin grafts also results in bcc - like changes ( callahan et al . , 2004 ) , which indicates that pathway activation is sufficient for tumorigenesis and provides compelling therapeutic targets in these tumors .
the first clues that the hh pathway could be targeted came from the observation that the plant - derived compound cyclopamine potently inhibits smo ( chen et al . , 2002b ) and has efficacy against hh - dependent tumors .
independent in vitro screens by several groups led to the discovery of other smo antagonists with distinct molecular structure , with one , vismodegib , recently approved for clinical use ( chen et al .
vismodegib was identified from a small molecule screen for hh pathway antagonists and achieved its final clinical form after chemical optimization for improved solubility , metabolic stability , and biodistribution ( robarge et al . , 2009 ) .
clinical trials using smo antagonists in patients reveal heterogeneous responses from bccs in different patient groups .
patients with basal cell nevus syndrome ( bcns ) carry a ptch1 mutation that predisposes them to develop hundreds of bccs with relatively little sun exposure . despite the high tumor burden
, smo blockade using vismodegib appears effective with a surprising 100% ( 38 of 38 ) response rate in patients ( tang et al . , 2012 ) .
although many lesions existed on each patient , no disease progression or acquired resistance developed during the treatment period ( mean of 8 mo ) , revealing a particularly sensitive tumor population with a slow rate of evolution .
vismodegib treatment appeared both tumoricidal and tumoristatic , as many of the tumors regrew with cessation of the drug .
phase i trials treating metastatic or locally advanced bcc found that only about half ( 19 of 33 patients ) displayed tumor regression ( von hoff et al .
2011 ) , despite having a similar bcc histology to the discussed bcns patients ( fig .
, patients in a phase ii clinical trial showed a response rate of 30% ( 10 of 33 ) in metastatic and 43% ( 27 of 63 ) in locally advanced bccs ( sekulic et al . , 2012 ) .
finally , more invasive solid tumors such as small cell lung or pancreatic cancers demonstrate little or no responsiveness in early phase clinical trials ( lorusso et al . , 2011 ) , despite an inhibition of hh pathway activity in uninvolved skin from the same patient .
although larger hh - dependent tumor studies need to be performed , early evidence supports the idea that more invasive tumor subtypes exhibit a much greater ability to resist smo blockade .
( a ) multiple untreated , but biopsy proven , bcc tumors in an individual with a genetic syndrome leading to shh overexpression .
( b ) vismodegib - treated tumors shrink , but several resistant tumors remain after 1 yr of drug treatment .
although one - third of advanced bcc tumors initially respond , persistent vismodegib therapy leads to significant secondary resistance ( chang and oro , 2012 ) .
of the 28 patients continuously treated with smo inhibitor , 21% developed at least one tumor regrowth while on the drug , with the mean time to detected regrowth at 56 wk ( fig .
these data indicate that more aggressive tumors display higher initial resistance and are more capable of evolving drug - independent growth characteristics .
one possibility for the differential response between tumors is that syndromic bccs are somehow different from sporadic bccs . arguing against this idea is the fact that 6 of the 28 patients were bcns patients that developed locally advanced disease , and 3 of these 6 patients acquired drug resistance during treatment .
it is possible that advanced bccs have higher mutation rates or more tumor cells , leading to a higher chance of resistant mutations that contribute to the ability of the tumor to evolve resistance .
the first clues that the hh pathway could be targeted came from the observation that the plant - derived compound cyclopamine potently inhibits smo ( chen et al . , 2002b ) and has efficacy against hh - dependent tumors .
independent in vitro screens by several groups led to the discovery of other smo antagonists with distinct molecular structure , with one , vismodegib , recently approved for clinical use ( chen et al .
vismodegib was identified from a small molecule screen for hh pathway antagonists and achieved its final clinical form after chemical optimization for improved solubility , metabolic stability , and biodistribution ( robarge et al . , 2009 ) .
clinical trials using smo antagonists in patients reveal heterogeneous responses from bccs in different patient groups .
patients with basal cell nevus syndrome ( bcns ) carry a ptch1 mutation that predisposes them to develop hundreds of bccs with relatively little sun exposure . despite the high tumor burden
, smo blockade using vismodegib appears effective with a surprising 100% ( 38 of 38 ) response rate in patients ( tang et al . , 2012 ) .
although many lesions existed on each patient , no disease progression or acquired resistance developed during the treatment period ( mean of 8 mo ) , revealing a particularly sensitive tumor population with a slow rate of evolution .
vismodegib treatment appeared both tumoricidal and tumoristatic , as many of the tumors regrew with cessation of the drug .
phase i trials treating metastatic or locally advanced bcc found that only about half ( 19 of 33 patients ) displayed tumor regression ( von hoff et al .
2011 ) , despite having a similar bcc histology to the discussed bcns patients ( fig .
, patients in a phase ii clinical trial showed a response rate of 30% ( 10 of 33 ) in metastatic and 43% ( 27 of 63 ) in locally advanced bccs ( sekulic et al . , 2012 ) .
finally , more invasive solid tumors such as small cell lung or pancreatic cancers demonstrate little or no responsiveness in early phase clinical trials ( lorusso et al . , 2011 ) , despite an inhibition of hh pathway activity in uninvolved skin from the same patient .
although larger hh - dependent tumor studies need to be performed , early evidence supports the idea that more invasive tumor subtypes exhibit a much greater ability to resist smo blockade .
( a ) multiple untreated , but biopsy proven , bcc tumors in an individual with a genetic syndrome leading to shh overexpression .
( b ) vismodegib - treated tumors shrink , but several resistant tumors remain after 1 yr of drug treatment .
although one - third of advanced bcc tumors initially respond , persistent vismodegib therapy leads to significant secondary resistance ( chang and oro , 2012 ) .
of the 28 patients continuously treated with smo inhibitor , 21% developed at least one tumor regrowth while on the drug , with the mean time to detected regrowth at 56 wk ( fig .
these data indicate that more aggressive tumors display higher initial resistance and are more capable of evolving drug - independent growth characteristics .
one possibility for the differential response between tumors is that syndromic bccs are somehow different from sporadic bccs . arguing against this idea is the fact that 6 of the 28 patients were bcns patients that developed locally advanced disease , and 3 of these 6 patients acquired drug resistance during treatment .
it is possible that advanced bccs have higher mutation rates or more tumor cells , leading to a higher chance of resistant mutations that contribute to the ability of the tumor to evolve resistance .
predicting tumor response to targeted therapies requires an understanding of the how mutations are generated and the evolutionary mechanisms used to bypass treatment .
many recent excellent reviews exist about the molecular basis of the mutator phenotype , where alterations in a mutator gene dramatically increase mutation rates at other loci ( loeb , 2011 ) .
however , a key question in drug - induced tumor resistance is : how do tumors bypass the smo blockade ?
advanced tumors can evolve resistance through pathway - dependent genetic mechanisms or through compensatory adaptation .
recent data indicate that pathway and compensatory intratumor heterogeneity exists at the time of diagnosis .
drug - resistant clones , initially present in low numbers , become the dominant clone as they gain growth advantage in the treated tumor ( diaz et al . ,
understanding common escape mechanisms may help prescreen patients to determine which targeted therapy or combination of targeted therapies is most likely to be effective .
pathway - dependent genetic alterations discovered in resistant tumors from patients and animal models directly affect hh pathway members .
initial studies in medulloblastoma suggest that vismodegib resistance stems from genetic alterations at the level of , or downstream from , smo .
drug interaction while maintaining hh pathway activation ( yauch et al . , 2009 ) .
other genetic alterations that lead to resistance come from gene duplications of gli2 or hh target gene cyclin d1 ( fig .
1 f ) that bypass the requirement of smo to inappropriately maintain or increase gli target gene induction ( buonamici et al . , 2010 ; dijkgraaf et al . ,
these mutations promote high hh pathway activation in the presence of smo antagonists and mediate resistant tumor growth .
compensatory alterations outside of the canonical hh pathway have also been found that mediate tumor resistance .
a compensatory alteration is one where elevated hh activation occurs in the absence of direct genetic mutation or copy number variation of hh pathway members , and is epigenetic in nature .
one example is the up - regulation of the phosphoinositide 3-kinase ( pi3k ) pathway ( buonamici et al . , 2010 ) . pi3k and
its downstream effectors were enriched in resistant versus sensitive medulloblastoma tumors , which suggests that pi3k signaling may in part promote tumor resistance by up - regulating hh pathway activation ( fig . 1 h ) . as pi3k
signaling can potentiate gli - dependent transcription induced by low levels of hh ( riob et al . ,
2006 ) , pi3k signaling may work best to promote tumor resistance when smo antagonists suppress hh pathway activation . in support of this hypothesis , pi3k or
pi3k - mtor inhibitors in combination with a smo antagonist can delay tumor resistance ( buonamici et al . ,
similarly , in esophageal cancers resistant to smo inhibitors , the activity of mtor pathway component s6 kinase 1 ( s6k1 ) was found to be elevated ( wang et al . ,
s6k1 phosphorylates gli1 , releasing gli1 from sufu inhibition to activate gli - dependent transcription ( fig .
interestingly , s6k1 is inappropriately activated in esophageal adenocarcinoma and some medulloblastomas ( dijkgraaf et al . , 2011 ; wang et al . , 2012 ) ,
predicting tumor response to targeted therapies requires an understanding of the how mutations are generated and the evolutionary mechanisms used to bypass treatment .
many recent excellent reviews exist about the molecular basis of the mutator phenotype , where alterations in a mutator gene dramatically increase mutation rates at other loci ( loeb , 2011 ) .
however , a key question in drug - induced tumor resistance is : how do tumors bypass the smo blockade ?
advanced tumors can evolve resistance through pathway - dependent genetic mechanisms or through compensatory adaptation .
recent data indicate that pathway and compensatory intratumor heterogeneity exists at the time of diagnosis .
drug - resistant clones , initially present in low numbers , become the dominant clone as they gain growth advantage in the treated tumor ( diaz et al . ,
understanding common escape mechanisms may help prescreen patients to determine which targeted therapy or combination of targeted therapies is most likely to be effective .
pathway - dependent genetic alterations discovered in resistant tumors from patients and animal models directly affect hh pathway members .
initial studies in medulloblastoma suggest that vismodegib resistance stems from genetic alterations at the level of , or downstream from , smo .
drug interaction while maintaining hh pathway activation ( yauch et al . , 2009 ) .
other genetic alterations that lead to resistance come from gene duplications of gli2 or hh target gene cyclin d1 ( fig .
1 f ) that bypass the requirement of smo to inappropriately maintain or increase gli target gene induction ( buonamici et al . , 2010 ; dijkgraaf et al . ,
these mutations promote high hh pathway activation in the presence of smo antagonists and mediate resistant tumor growth .
compensatory alterations outside of the canonical hh pathway have also been found that mediate tumor resistance .
a compensatory alteration is one where elevated hh activation occurs in the absence of direct genetic mutation or copy number variation of hh pathway members , and is epigenetic in nature .
one example is the up - regulation of the phosphoinositide 3-kinase ( pi3k ) pathway ( buonamici et al . , 2010 ) . pi3k and
its downstream effectors were enriched in resistant versus sensitive medulloblastoma tumors , which suggests that pi3k signaling may in part promote tumor resistance by up - regulating hh pathway activation ( fig . 1 h ) . as pi3k
signaling can potentiate gli - dependent transcription induced by low levels of hh ( riob et al . ,
2006 ) , pi3k signaling may work best to promote tumor resistance when smo antagonists suppress hh pathway activation . in support of this hypothesis , pi3k or
pi3k - mtor inhibitors in combination with a smo antagonist can delay tumor resistance ( buonamici et al . ,
similarly , in esophageal cancers resistant to smo inhibitors , the activity of mtor pathway component s6 kinase 1 ( s6k1 ) was found to be elevated ( wang et al . ,
s6k1 phosphorylates gli1 , releasing gli1 from sufu inhibition to activate gli - dependent transcription ( fig .
interestingly , s6k1 is inappropriately activated in esophageal adenocarcinoma and some medulloblastomas ( dijkgraaf et al . , 2011 ; wang et al . , 2012 ) ,
developing effective targeted therapies to dispatch tumors before they evolve resistance requires knowledge of available escape pathways .
this is especially critical given that resistant clones are likely present in small numbers at the time of treatment initiation . given the pathway and compensatory alterations in hh - dependent tumors thus far , dual targeting of the most downstream component of the pathway , and the compensatory pathways , will likely generate optimal therapies .
gli transcription factors ultimately transduce the signal from the hh ligand ; moreover , escape pathways that bypass smo still activate gli .
targeting gli directly or the signaling components that activate gli could prove quite successful as the next level of therapy .
several gli inhibitors have been identified that have impressive efficacy . through a screen of the national institutes of health compound library , molecules such as gant58 and gant61 were found to block gli transcriptional activity ( lauth et al . , 2007 ) .
these drugs inhibit hh signaling with a similar ic50 to cyclopamine and suppress gli1-positive human prostate cancer xenografts
. moreover , similar screens using a library of previously fda - approved drugs identified arsenic trioxide ( ato ) as a gli antagonist ( kim et al . , 2010 ) .
ato blocks accumulation of gli2 to primary cilia , with longer incubation times reducing steady - state gli2 protein levels , resulting in suppression of medulloblastoma growth in mouse models .
as ato is already in clinical use for acute promyelocytic leukemia , this may be a useful therapy for resistant bccs in the near future .
identifying and targeting modulators of gli activity may also show promise in preventing or treating resistant tumors .
new pathways that regulate gli such as the aforementioned s6k1 may hold the key to delay tumor resistance .
because s6k1 appears to regulate gli1 activity downstream of mtor , and smo inhibitors regulate gli activity and nuclear localization , combination therapy may prevent tumor cells containing this compensatory alteration from growing .
indeed , in vitro data from esophageal adenocarcinoma xenografts suggest that combination therapy with mtor inhibitors and vismodegib work synergistically ( wang et al . , 2012 ) .
these data provide strong preclinical support for the use of combined therapy to delay growth of smo antagonist  resistant tumors . the blueprint to develop the first hh pathway inhibitors came from impressive efforts from the hh and cancer communities , beginning with identification of hh pathway components and their roles in cancer and continuing with intense screening and medicinal chemistry that refined drug targets for optimal human use .
the challenge for the future is to better understand common pathway - dependent genetic and compensatory escape pathways that evolve from clonal populations within tumors and design combination therapies to block them before further evolution takes place . through improved genomics , cell biology , and medicinal chemistry
developing effective targeted therapies to dispatch tumors before they evolve resistance requires knowledge of available escape pathways .
this is especially critical given that resistant clones are likely present in small numbers at the time of treatment initiation . given the pathway and compensatory alterations in hh - dependent tumors thus far , dual targeting of the most downstream component of the pathway , and the compensatory pathways , will likely generate optimal therapies .
gli transcription factors ultimately transduce the signal from the hh ligand ; moreover , escape pathways that bypass smo still activate gli .
targeting gli directly or the signaling components that activate gli could prove quite successful as the next level of therapy .
several gli inhibitors have been identified that have impressive efficacy . through a screen of the national institutes of health compound library , molecules such as gant58 and gant61 were found to block gli transcriptional activity ( lauth et al . , 2007 ) .
these drugs inhibit hh signaling with a similar ic50 to cyclopamine and suppress gli1-positive human prostate cancer xenografts
. moreover , similar screens using a library of previously fda - approved drugs identified arsenic trioxide ( ato ) as a gli antagonist ( kim et al . , 2010 ) .
ato blocks accumulation of gli2 to primary cilia , with longer incubation times reducing steady - state gli2 protein levels , resulting in suppression of medulloblastoma growth in mouse models .
as ato is already in clinical use for acute promyelocytic leukemia , this may be a useful therapy for resistant bccs in the near future .
identifying and targeting modulators of gli activity may also show promise in preventing or treating resistant tumors .
new pathways that regulate gli such as the aforementioned s6k1 may hold the key to delay tumor resistance .
because s6k1 appears to regulate gli1 activity downstream of mtor , and smo inhibitors regulate gli activity and nuclear localization , combination therapy may prevent tumor cells containing this compensatory alteration from growing .
indeed , in vitro data from esophageal adenocarcinoma xenografts suggest that combination therapy with mtor inhibitors and vismodegib work synergistically ( wang et al . , 2012 ) .
these data provide strong preclinical support for the use of combined therapy to delay growth of smo antagonist  resistant tumors . the blueprint to develop the first hh pathway inhibitors came from impressive efforts from the hh and cancer communities , beginning with identification of hh pathway components and their roles in cancer and continuing with intense screening and medicinal chemistry that refined drug targets for optimal human use .
the challenge for the future is to better understand common pathway - dependent genetic and compensatory escape pathways that evolve from clonal populations within tumors and design combination therapies to block them before further evolution takes place . through improved genomics , cell biology , and medicinal chemistry
 dependence of basal cell carcinomas and medulloblastomas on the hedgehog pathway provides an opportunity for targeted or  
 personalized  therapy . 
 the recent effectiveness and fda approval of the first smoothened inhibitors validates this class of agents , but has revealed drug - resistant tumor variants that bypass smoothened inhibition . here , 
 we summarize the effectiveness of hedgehog pathway inhibitors and highlight promising areas for the development of next generation drug antagonists for hedgehog - dependent cancers . 
A compelling connection to human cancer
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The first Smo inhibitor: How effective is it?
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Pathway and compensatory tumor evolution in treated patients
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Racing against tumor evolution