in 1980 , hedgehog signaling was first discovered in the fruit fly by nusslein - volhard , and has since been found in vertebrates within various organs . hedgehog signaling , a pathway characterized by being conserved but considerably multifunctional , is involved in a variety of developmental and physiological processes , such as body axis formation , angiogenesis , and stem cell homeostasis . as a result , the developing tissues grow into the correct size with the appropriate cell types , orientation , and vascularization . according to the world cancer report in 2012 , pancreatic cancer was ranked as the seventh most common cause of cancer deaths , with 330,000 deaths globally and a 5-year survival of less than 5% . pancreatic cancer cells exhibit tenacious growth , early dissemination , metastatic ability , and resistance to radiotherapy and chemotherapy , all of which contribute to high mortality . without proper and early diagnosis , delayed detection is common . in this case , most patients are diagnosed with end - stage pancreatic carcinoma . thus , only 10%15% patients are able to receive surgery , even though an operation is still the most valid therapeutic method ; the 5-year survival of these patients is approximately 10% . the patients who are unable to undergo surgery will inevitably suffer through chemotherapy and radiotherapy . the standard remedy for pancreatic cancer established by burris et , the significantly improved 0.3-month survival advantage seems to have no obvious effect on clinical treatment . currently , the underlying mechanism of hedgehog signaling in carcinoma is being increasingly studied , as such a somber condition as pancreatic cancer warrants the development of novel and effective methods . pancreatic tissue fibrosis is a terminal and distinguishing feature of pathological changes with diverse means of inflicting harm . the formation of pancreatic fibrosis is a complicated and long - term process in which multiple factors interact with each other . injuries ( apoptosis and necrosis ) of the pancreas can induce the synthesis and release of proinflammatory factors , chemokines and growth factors such as pdgf , tgf-1 , and angiotensin ii , resulting in the activation of pancreatic stellate cells ( pscs ) and the accumulation of myofibroblasts . myofibroblasts are terminally differentiated cells that are responsible for the synthesis and deposition of extracellular matrix ( ecm ) components such as type i and iii collagens . if repair mechanisms are disrupted or ineffective , excessive deposition of ecm components will form a barrier around the original pathological injury , leading to the intensive resistance to radiotherapy and chemotherapy . inhibition of hedgehog signaling can reduce or even reverse pscs activation , leading to improved outcomes in chronic pancreatitis . for example , resveratrol , a botanical compound derived mainly from the skins of red grapes , may have antifibrotic effects on the pancreas by antagonizing the hedgehog pathway . therefore , screening of highly effective pharmaceutical agents to inhibit the activation of hedgehog signaling provides a great opportunity for the development of antifibrotic drugs . the hedgehog signaling pathway is classified into 2 modalities : canonical and noncanonical . noncanonical hedgehog signaling refers to hedgehog signaling receptor dependent signals that do not operate via gli or smo . noncanonical hedgehog signaling is divided into 2 types : type i acts through ptch , while type ii acts through smo without being regulated by gli . in total , hedgehog signaling molecules include 3 ligands ( sonic hedgehog ( shh ) , indian hedgehog ( ihh ) , and desert hedgehog ( dhh ) ) , 2 receptors ( ptch1 and ptch2 ) , a signal transducer smoothened ( smo ) , and 3 transcription factors ( gli1 , gli2 , and gli3 ) . , the hedgehog ligand acts as the initiating event in a signaling cascade . when the signaling pathway is activated , hedgehog ligand undergoes autoprocessing and lipid modification reactions to generate the hedgehog signaling peptide with n - terminal palmitoylation and c - terminal cholesterylation , and thereby activates itself . mature hedgehog peptide with lipid modifications will be released from secreting cells by multitransmembrane transporter - like proteins that are dispatched and transduced to target cells through the multiple - transmembrane receptor ptch . ptch is encoded by the patched gene and is a 12-transmembrane protein that acts as the receptor for hedgehog signaling.patched is a tumor suppressor gene and mutations in this gene can lead to gorlin syndrome , medulloblastoma , and esophageal squamous cancer . ptch1 , which is confined to target cells , is upregulated in response to hedgehog - signaling proteins . it is the subtype that is definitively involved in the activation of hedgehog signaling . the transcription of ptch2 is independent of pathway activation and is coexpressed with hedgehog proteins . goodrich et al found in ptch1 knockout mice that the inhibition of smo activity was abolished . further studies confirmed that ptch inhibits smo activity when hedgehog ligands are absent . when hedgehog peptides are released and activate the hedgehog receptors of target cells , ptch activity is suppressed , leading to smo translocation to the plasma membrane , interaction with cos2 , and increased autophosphorylation . as previously reported , the small molecule cyclopamine targets the smo protein to inhibit hedgehog signaling . in drosophilae , smo directly recruits the cytoplasmic complex cos2/ci / fu through cos2 and then influences their activity . in the absence of smo , the complex cos2/ci / fu phosphorylates and binds to the zinc finger - like transcription factor gli . in summary , the hedgehog ligand protein spindles ptch , which is bound to the ciliary , thereby inducing the release of smo ; then , the gli protein is released from smo and is translocated to the nucleus where it functions as a transcriptional activator . only the repressed form of gli the aberrant expression of gli1 or the dominant activating mutant gli2 in keratinocytes regulates overlapping transcriptional processes of these 2 proteins ( figure 1 ) . a , in the absence of hedgehog ligand ( e.g. , shh ) , ptch inhibits smo from reaching the plasma membrane . in this case , the microtubule - associated cos2fu sufu complex can bind full - length gli , which can be phosphorylated by glycogen synthase kinase-3 ( gsk-3 ) , protein kinase a ( pka ) , and casein kinase 1 ( ck1 ) . phosphorylated gli is cleaved to an n - terminal form and then will translocate the nucleus to suppress transcription . b , in the presence of hedgehog ligand , ptch activity is suppressed , and thereby smo translocates to the plasma membrane and interacts with cos2 . in this state , the cos2fu  sufu complex can not bind gli , and gli is able to enter the nucleus and induce transcription of target genes . in the blue and orange frames blue frame : type i requires only binding of a hedgehog isoform to ptch and is mediated by novel functions of ptch unrelated to smo repression , and it is by definition insensitive to smo modulators . orange frame : type ii is dependent on smo and in some cases it has been shown to rely on signaling through gli proteins , and it is both mimicked by smo agonists and inhibited by smo antagonists . in contrast to the canonical pathway referred to above , studies have demonstrated that not all hedgehog signaling proceeds through gli activation ; these subtypes of gli - independent pathways were named noncanonical hedgehog signaling . the binding of a hedgehog isoform to ptch is required in type i noncanonical hedgehog signaling , which is mediated by the novel functions of ptch that are unrelated to smo repression and are thus insensitive to modulators of smo . in the absence of a hedgehog ligand , several studies corroborate the hypothesis that the proapoptotic effect of ptch1 is exerted through a type i noncanonical pathway . for example , the in vitro or in vivo disruption of detectable canonical hedgehog signaling , the absence of an effect of smo antagonists to prevent the shh - dependent reduction in caspase-3 activity and the inability of the smo agonist to mimic the antiapoptotic effect of shh , and ptch1 regulation of the cell cycle through cyclin b1 in a smo- and gli - independent manner . compared with type i noncanonical hedgehog signaling , type ii signaling is defined by smo - dependent and gli - independent cascades . hedgehog signaling responds to smo agonists that are mediated through the activation of small gtpases to elicit cellular responses . bijlsma et al found that shh - induced fibroblast migration was smo - dependent but gli - independent . in chinchilla study , hedgehog proteins in endothelial cells promoted actin stress fiber formation and endothelial cell tubulogenesis in a smo - dependent manner . in axonal development , shh signals stimulated the activity of src family kinase members in a smo - dependent manner . in a later study , a novel signaling cascade operating through sh3 domain - containing proteins was found to be directly stimulated by hedgehog ligands ( figure 1 ) . in humans , the pancreas is located in the abdominal cavity behind the stomach and acts as an endocrine and exocrine gland for the digestive and endocrine systems . hedgehog signaling influences the development of both endocrine and exocrine functions from the newborn to the mature adult . staining of embryos reveals that shh signaling is expressed throughout the gut endoderm , with the exception of the pancreatic bud endoderm . during embryogenesis , the pancreas develops from the pancreas bud of the end endodermal foregut and its surrounding mesoderm . the function of pancreatic cells emerges from common precursors present in the early gut endoderm . pancreatic bud endoderm expresses high levels of the homeodomain protein ipf1/pdx1 ( insulin promoter factor 1/pancreatic and duodenal homeobox 1 ) , an essential regulator of early pancreatic development . when pancreatic explants were exposed to shh , ipf1/pdx1 induced constitutive shh overexpression in the pancreatic bud , thus promoting pancreatic mesoderm differentiation into smooth muscle and interstitial cajal cells , which are typical of the developing intestine rather than the pancreatic mesenchyme and spleen . inhibition of hedgehog signaling with the specific inhibitor cyclopamine would induce a variety of changes in the pancreas , for example , gastric explants expressing endocrine pancreatic markers such as insulin and glucagon , an increase in islet cell number and size and the high level expression of ipf1 . the experiment conducted by diiorio in zebrafish clarified that gene mutation of shh and the relative signaling pathway had the specific dependence with endocrine function . these mutants confirmed that during gastrulation , the transient shh signaling induced the pancreatic endoderm to differentiate into the islet tissue subsequently . in the later development , a second hedgehog - dependent activity appeared which was similar to role of shh in the foregut endoderm assisting the localization of pancreas . the biological function of hedgehog signaling in the pancreatic epithelium remains controversial , while activation or inhibition of hedgehog signaling pathway only has slight effect on epithelial development . it suggests that hedgehog pathway might mainly focus in pancreatic mesenchyme . during embryo development , beta - cell was transiently delayed due to the pdx1-driven loss of smo that lau and hebrok discovered in pancreatic epithelial progenitor cells of pdx1/smo mice . after given birth , beta - cell numbers would restore ; however they were dysfunctional . in this case , the phenomenon ( reduced insulin secretion , slight insulin - dependent diabetes , and enhanced insulin sensitivity ) occurred . the primary cilium that was structurally required as signaling downstream of smo had close relationship with the mild responsiveness to the activation of hedgehog signaling in pancreatic epithelial cells . overactivation of hedgehog pathway in mature epithelial cell specifically with resection of primary cilium caused the following results that pancreas tissue dedifferentiated , markers of progenitor cells reexpressed , and endocrine area decreased . pscs are myofibroblast - like cells that can switch between quiescent and activated phenotypes , such as hepatic stellate cells , and reside in exocrine areas of the pancreas . when pathogens lead to pancreatic fibrosis or carcinoma , the sensitive signaling of the hedgehog pathway is easily activated . activated pscs will migrate to the injured location and participate in tissue repair activities by secreting ecm components . research by shinozaki et al revealed in an in vitro study that exogenous ihh protein could enhance the migrational ability of pscs by increasing membrane type-1 matrix metalloproteinase on the plasma membrane , while gli1 negatively regulated ihh stimulated pscs migration by inhibiting active mt1-mmp . myofibroblasts were detected in capan-2 tumors , thus supporting the hypothesis that these represented pancreatic stellate cells within the tumor and proliferated in response to shh stimulation . jennifer and colleagues stimulated pscs with recombinant shh for 24  hours at 1 and 10  g / ml , and observed an increase in the mesenchymal markers sma , vimentin and desmin and a decrease in the epithelial marker ck19 . this was the first study to show that shh induces the differentiation of pscs into myofibroblasts . however , in the stroma , tumor cells overexpressing shh led to the consequences that activated the hedgehog signaling pathway in pscs instead of themselves . activated pscs were crucial in enhancing neural invasion of pancreatic cancer along axons and nerve growth of cancer cell colonies that assisted pancreatic cancer cell migration . in vivo pancreatic cancer model , paracrine shh activated the pscs and finally resulted in the tumor cell invasion of the trunk , sciatic nerve dysfunction , and the growth and metastasis of the orthotopic xenograft tumor , while the inhibitor cyclopamine could impede these pathological changes . expression of hedgehog signaling pathway components revealed that smo and gli were primarily observed in stromal - derived pscs , whereas the ligands shh and ihh were limited to pancreatic tumor cells . the development of pdac tumors was not affected when smo was genetically ablated in the pancreatic epithelium of pdac - susceptible mice . treatment of pscs with shh and ihh activated the hedgehog signaling pathway ( the upregulation of gli mrna supports this finding ) , and stimulated the proliferation of pscs . hedgehog signaling ligands secreted from neighboring cancer cells stimulated pscs to produce soluble factors that then fed back on cancer cells to promote their activity . when the pancreas suffers injuries , pscs undergo morphological and functional changes to become activated to express -smooth muscle actin . they will proliferate , migrate , and secrete ecm proteins , thus leading to pancreatic fibrosis . hedgehog signaling induces progressive pancreatic fibrosis intermingled with proliferating ductal structures that are accompanied by the destruction of acinar structures . paracrine hedgehog signaling activates myofibroblasts and leads to their proliferation , and it shows the restricted expression of hedgehog signaling downstream components including ptch , smo , and gli1/2 in hedgehog - responsive cells , while hedgehog signaling ligands induce matrix metalloproteinases ( mmps ) in all hedgehog - responsive cells , as shown in figure 2 . the hedgehog signaling pathway can be expressed in the embryonic mouse pancreas and is also observed in the adult mouse pancreas . because ihh expression is elevated in the pancreas of patients with chronic pancreatitis , ihh is thought to participate in chronic pancreatic injury , especially pancreatic fibrosis . islet cells of tissues undergoing chronic pancreatitis exhibit an abnormal localization pattern for ihh . as a signal transducer , smo is upregulated in cancer - associated stromal fibroblasts , and the expressed smo could transduce the shh to activate gli1 expression . however , ihh does not directly activate pscs nor stimulate psc proliferation ; in other words , the parameter of pscs transformation ( expression of collagen-1 or alpha - smooth muscle actin ) stays unresponsive to ihh . ihh has a powerful effect on psc migration not only in chemotactic manner but also in chemokinetic way . when target cells get stimulated and need to migrate , ihh raises the level of membrane - type 1 matrix metalloproteinase ( mt1-mmp ) and shifts them onto the plasma membrane from original location . according to the result from satoshi shinazaki and his colleagues they applied adenovirus to cause gli1 overexpression and rna interference technique to reduce gli1 expression . the results that overexpression of gli1 nevertheless blocks mt1-mmp localization on the plasma membrane and pscs migration while rna interference - mediated knockdown of gli1 augments pscs migration indicated that there was a negative feedback loop in the relationship between ihh and gli1 . during in vitro wound healing , shh increases the ability of myofibroblasts to migrate into a wounded area as a monolayer . shh significantly increases the ability of myofibroblasts to recover from the wound and promotes the migration and invasion of human pancreatic myofibroblasts in vitro . there are 3 hedgehog signaling coreceptors , gas1 , boc , and cdon , which are cell - surface - associated proteins that act as pathway activators and are expressed in cancer - associated fibroblasts . the deletion of 2 coreceptors ( gas1 and boc ) in fibroblasts leads to a reduction in hedgehog signaling responsiveness . additionally , these fibroblasts promote greater tumor growth and tumor - associated vascularity . the deletion of all 3 coreceptors ( gas1 , boc , and cdon ) results in the almost complete abrogation of hedgehog signaling and a corresponding failure to promote tumorigenesis and angiogenesis . in general , the study by mathew et al identified a role for hedgehog dosage in the promotion of pancreatic cancer and interpreted the clinical failure of blocking the hedgehog signaling pathway as a therapeutic approach in pancreatic cancer . referring to tumorigenesis , hedegehog signaling has the nonnegligible relationship . the role of hedgehog signaling relating to carcinoma was first identified in patients with gorlin syndrome caused by mutation of ptch . once loss - of - function mutations in ptch or mutations in smo , it leads to sustained activation of hedgehog pathway . overexpression of gli1 and hedgehog proteins is associated with a variety of cancers and implicated in the onset of pancreatic ductal neoplasia and the maintenance of advanced cancers . not as we expected , in pancreatic cancer , ligand - dependent activation of hedgehog signaling instead of genomic mutation was reported and overexpression of shh was considered adequate to trigger the initiation of pancreatic cancer . as the vital effect of hedgehog signaling on pancreatic cancer , many other factors may interact with it to influence the development of the malignant disease . perineural invasion is a common pathologic feature in pancreatic cancer by which cancer cells invade the peripheral nerves and are disseminated . several studies showed that stromal pscs may play a regulatory role in the interaction between cancer cells and nerves . specific cell types and tissues that release bioactive shh from pancreatic cancer cells can produce heparan sulfate ( hs ) . according to in vivo knockdown and in vitro cell culture studies , glypican hs proteoglycans release bioactive shh morphogens from the surface of transfected bosc23 cells through hs chains in a cell autonomous manner . hs specifically modifies shh processing on the cell surface , and purified glycosaminoglycans enhance the proteolytic removal of n- and c - terminal shh peptides under cell - free conditions . the overexpression of shh in tumor cells activates the hedgehog signaling pathway in pscs in the stroma rather than activating tumor cells directly to augment the output of mmp2 , mmp9 , and ngf . these activated pscs and associated molecules promote pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies . coimplantation of pscs activated by paracrine shh induces tumor cell invasion of the trunk , stimulates nerve dysfunction , and promotes orthotropic xenograft tumor growth , metastasis , and perineural invasion in vivo . in recent years , several experiments proved that in both vivo and vitro , the hippo pathway exerted regulator ability of cell density . when the cell density exceeded the threshold , the hippo cascade would be turned on and lead to the activation of large tumor suppressor ( lats ) kinases , finally causing the phosphorylation of yes - associated protein and its paralog taz ( yap / taz ) . in cell culture , the activity of hedgehog signaling is dependent on cell - to - cell contact . when the cell intimately contacted with each other or the intensity reached high enough , hedgehog pathway activity would be regulated to higher level . adenovirus - mediated yap overexpression nevertheless prevents hedgehog signaling while the knockdown of yap expression rna interference augments hedgehog / gli activity . however , hedgehog signaling enhances the post - transcription of yap to promote its activity . however , in human and mouse pancreatic cancers , low hedgehog signaling pathway activity accompanies strong nuclear yap immunoreactivity . but tumor could utilize both ocogenic pathway simultaneously on the condition there exist protease - activated receptors ( pars ) which has the potential ability to override the hippo / hedgehog pathway . as a converse effect of yap , brd4 , a regulator of epigenetic proteins that can activate shh members in a ligand - independent manner in pdac cells , has recently appeared as an alternative therapeutic strategy . suppression of brd4 damaged pdac cell viability and proliferation and negatively influenced the tumor growth rate . combination treatment of gemcitabine and brd4 silencing has a synergistic effect on chemotherapeutic efficacy and significantly promoted apoptosis in the panc-1 and miapaca-2 cell lines . its activity in pancreatic fibroblasts leads to the expression of il-6 , an inflammatory cytokine that activates stat3 in pancreatic cancer cells . improper stromal remodeling and persistence of the inflammatory infiltrate in pancreatic tumorigenesis is a consequence of the deletion of gli1 , while partial loss of gli1 affects fibrogenesis and the recruitment of immune cells . il-6 , mil-8 , mcp-1 , and m - csf ( csf1 ) , as a subset of cytokines , potentially direct gli1 target genes to mediate this phenomenon . in recent years , noncoding rna has become a popular in many fields . micrornas ( mirnas ) are a class of small noncoding rnas that play important roles in carcinogenesis . smo is a direct target of mir-125b , mir-193b , mir-324 - 5p mir-326 , and mir-338 - 3p . downregulation of mir-125b , mir-193b , mir-324 - 5p , mir-326 , and mir-338 - 3p in human cancers derepress smo and promote tumor proliferation and invasion through aberrant hedgehog signaling . mir-324 - 5p downregulation is caused by deletion of the mir-324 - 5p gene in a high percentage of mbs as a consequence of the loss of chromosome 17p . deletion of chromosome 17p leads to the loss of certain genes , thus contributing to hedgehog induced tumorigenesis . the levels of tumor suppressor mir - let7b , which targets several genes ( k - ras , muc4 , ncoa3 , hmga2 , tgfr1 , and stat3 phosphorylation ) involved in pdac pathogenesis , are down - regulated . kumar et al found that the combination therapy of mir - let7b and gdc-0449 effectively inhibited tumor growth when injected to athymic nude mice bearing ectopic tumors generated using mia paca-2 cells compared with micelles carrying gdc-0449 or mir - let7b alone . gain - of - function and loss - of - function experiments show that a pro - oncogenic function of mir-212 exists in pdac . this suggests that mir-212 could facilitate pdac progression and metastasis by targeting ptch1 , implicating a novel mechanism for the progression of pdac . mirna is not the only relevant nucleic acid , as some discoveries in dna have been made as well . twenty - five members have been reported to belong to the s100 family in humans . twenty - one of them are known as the epidermal differentiation complex ( edc ) involved in epithelial - derived cell differentiation , which are coded by genes clustered at chromosome locus 1q21 . s100a2 , s100a4 , s100a6 , s100a11 , and s100a14 from the s100 gene family are found to be significantly down - regulated due to gli1 knockdown . s100a4 and vimentin genes are up - regulated significantly by increased shh / gli1 expression , while e - cadherin is significantly reduced at the same time . migration of pancreatic cancer cells is increased significantly with dose - dependent increases in gli1 expression . silencing of s100a4 significantly reversed the response of pancreatic cancer cells induced by l - shh transduction . hedgehog signaling pathway inhibitors accelerate rather than delay the progression of oncogenic kras - driven disease . this finding was substantiated in 3 distinct genetically engineered mouse models used to study the effects of genetic or pharmacologic inhibition of the hedgehog signaling pathway activity . the balance between epithelial and stromal elements is notably influenced by pharmacologic inhibition of hedgehog , thus suppressing stromal desmoplasia . in contrast , the activation of this pathway using a small molecule agonist causes stromal hyperplasia and reduces epithelial proliferation . the stromal response to hedgehog signaling is protective against pdac , and the pharmacologic activation of the pathway can slow tumorigenesis . thus , it may offer an explanation for the failure of hedgehog inhibitors in clinical trials and may offer new insights into novel therapeutic interventions . pancreatic cancer is a highly aggressive carcinoma with an ultrahigh case fatality rate due to poor diagnosis and the lack of proper therapies . considering the unique role hedgehog signaling plays in pancreatic cancer , many strategies are focused on utilizing this pathway as a diagnostic tool for pancreatic carcinoma . clinical studies showed that the levels of shh in human blood were lower in patients with pancreatitis and pancreatic cancer compared with healthy individuals . reduced plasma levels of hedgehog were found in pancreatic cancer patients , but they were insufficient alone to predict pancreatic cancer . the epithelial - to - mesenchymal transition ( emt ) is a common phenomenon in various cancers . hedgehog signaling controls emt , enhances cell proliferation in an mapk- and pi3-kinase - dependent manner , decreases apoptosis through the regulation of bcl-2 and bcl - x , and induces the proliferation of cancer stem cells . silencing of gli1 alone without external stimulus had no effect on emt , but inhibited tgf-1 and egf - induced emt . it performs a protumor role in the aggressive invasion of pscs by promoting tgf-1 and egf - induced emt . shh and the clinical outcome in esophageal cancer demonstrated that gli1 was a strong and independent prognostic factor for a poor outcome . furthermore , gli1 and shh are independent of the major clinical features known to influence prognosis and could serve as an adjunct to current staging systems . importantly , no interaction was observed among gli1 , shh , and adjuvant treatment activity , ensuring that this confounding factor will not affect the relationship between gli1 and shh . in resected pancreatic ductal adenocarcinoma ( pdac ) , gli1 expression is essential for pdac cell survival by facilitating the migration and invasion of cells by promoting emt . lower expression of gli1 or shh in tumor cells contributed to longer disease free survival ( dfs ) and overall survival ( os ) times . the combination of shh and gli1 levels is thought to be the most significant predictor for overall survival of os . previous studies demonstrated that activation of both the nf-b and hedgehog signaling pathways played prominent roles in the initiation and progression of pancreatic cancer by acting not only in the cancer cell itself ( autocrine ) but also in stromal cells ( paracrine ) , while the expression of either nuclear or cytoplasmic nf-b was a poor prognostic factor for pancreatic cancer . nuclear expression of either gli1 or nf-b ( rela / p65 ) is associated with poor overall survival in pancreatic cancer patients , and nuclear expression of nf-b was the only significant prognostic factor according to multivariate analysis . in conclusion , nf-b expression , mmp9 expression , and gli1 expression may have prognostic significance in advanced pancreatic cancer and could be used as biomarkers to guide therapies for pancreatic adenocarcinoma in the future . noncanonical hedgehog signaling refers to hedgehog signaling receptor dependent signals that do not operate via gli or smo . noncanonical hedgehog signaling is divided into 2 types : type i acts through ptch , while type ii acts through smo without being regulated by gli . in total , hedgehog signaling molecules include 3 ligands ( sonic hedgehog ( shh ) , indian hedgehog ( ihh ) , and desert hedgehog ( dhh ) ) , 2 receptors ( ptch1 and ptch2 ) , a signal transducer smoothened ( smo ) , and 3 transcription factors ( gli1 , gli2 , and gli3 ) . , the hedgehog ligand acts as the initiating event in a signaling cascade . when the signaling pathway is activated , hedgehog ligand undergoes autoprocessing and lipid modification reactions to generate the hedgehog signaling peptide with n - terminal palmitoylation and c - terminal cholesterylation , and thereby activates itself . mature hedgehog peptide with lipid modifications will be released from secreting cells by multitransmembrane transporter - like proteins that are dispatched and transduced to target cells through the multiple - transmembrane receptor ptch . ptch is encoded by the patched gene and is a 12-transmembrane protein that acts as the receptor for hedgehog signaling.patched is a tumor suppressor gene and mutations in this gene can lead to gorlin syndrome , medulloblastoma , and esophageal squamous cancer . ptch1 , which is confined to target cells , is upregulated in response to hedgehog - signaling proteins . it is the subtype that is definitively involved in the activation of hedgehog signaling . the transcription of ptch2 is independent of pathway activation and is coexpressed with hedgehog proteins . goodrich et al found in ptch1 knockout mice that the inhibition of smo activity was abolished . further studies confirmed that ptch inhibits smo activity when hedgehog ligands are absent . when hedgehog peptides are released and activate the hedgehog receptors of target cells , ptch activity is suppressed , leading to smo translocation to the plasma membrane , interaction with cos2 , and increased autophosphorylation . as previously reported , the small molecule cyclopamine targets the smo protein to inhibit hedgehog signaling . in drosophilae , smo directly recruits the cytoplasmic complex cos2/ci / fu through cos2 and then influences their activity . in the absence of smo , the complex cos2/ci / fu phosphorylates and binds to the zinc finger - like transcription factor gli . in summary , the hedgehog ligand protein spindles ptch , which is bound to the ciliary , thereby inducing the release of smo ; then , the gli protein is released from smo and is translocated to the nucleus where it functions as a transcriptional activator . only the repressed form of gli the aberrant expression of gli1 or the dominant activating mutant gli2 in keratinocytes regulates overlapping transcriptional processes of these 2 proteins ( figure 1 ) . a , in the absence of hedgehog ligand ( e.g. , shh ) , ptch inhibits smo from reaching the plasma membrane . in this case , the microtubule - associated cos2fu  sufu complex can bind full - length gli , which can be phosphorylated by glycogen synthase kinase-3 ( gsk-3 ) , protein kinase a ( pka ) , and casein kinase 1 ( ck1 ) . phosphorylated gli is cleaved to an n - terminal form and then will translocate the nucleus to suppress transcription . b , in the presence of hedgehog ligand , ptch activity is suppressed , and thereby smo translocates to the plasma membrane and interacts with cos2 . in this state , the cos2fu  sufu complex can not bind gli , and gli is able to enter the nucleus and induce transcription of target genes . in the blue and orange frames there are 2 types of noncanonical hedgehog signaling . blue frame : type i requires only binding of a hedgehog isoform to ptch and is mediated by novel functions of ptch unrelated to smo repression , and it is by definition insensitive to smo modulators . orange frame : type ii is dependent on smo and in some cases it has been shown to rely on signaling through gli proteins , and it is both mimicked by smo agonists and inhibited by smo antagonists . in contrast to the canonical pathway referred to above , studies have demonstrated that not all hedgehog signaling proceeds through gli activation ; these subtypes of gli - independent pathways were named noncanonical hedgehog signaling . the binding of a hedgehog isoform to ptch is required in type i noncanonical hedgehog signaling , which is mediated by the novel functions of ptch that are unrelated to smo repression and are thus insensitive to modulators of smo . in the absence of a hedgehog ligand , several studies corroborate the hypothesis that the proapoptotic effect of ptch1 is exerted through a type i noncanonical pathway . for example , the in vitro or in vivo disruption of detectable canonical hedgehog signaling , the absence of an effect of smo antagonists to prevent the shh - dependent reduction in caspase-3 activity and the inability of the smo agonist to mimic the antiapoptotic effect of shh , and ptch1 regulation of the cell cycle through cyclin b1 in a smo- and gli - independent manner . compared with type i noncanonical hedgehog signaling , type ii signaling is defined by smo - dependent and gli - independent cascades . hedgehog signaling responds to smo agonists that are mediated through the activation of small gtpases to elicit cellular responses . bijlsma et al found that shh - induced fibroblast migration was smo - dependent but gli - independent . in chinchilla study , hedgehog proteins in endothelial cells promoted actin stress fiber formation and endothelial cell tubulogenesis in a smo - dependent manner . in axonal development , shh signals stimulated the activity of src family kinase members in a smo - dependent manner . in a later study , a novel signaling cascade operating through sh3 domain - containing proteins was found to be directly stimulated by hedgehog ligands ( figure 1 ) . in humans , the pancreas is located in the abdominal cavity behind the stomach and acts as an endocrine and exocrine gland for the digestive and endocrine systems . hedgehog signaling influences the development of both endocrine and exocrine functions from the newborn to the mature adult . staining of embryos reveals that shh signaling is expressed throughout the gut endoderm , with the exception of the pancreatic bud endoderm . during embryogenesis , the pancreas develops from the pancreas bud of the end endodermal foregut and its surrounding mesoderm . the function of pancreatic cells emerges from common precursors present in the early gut endoderm . pancreatic bud endoderm expresses high levels of the homeodomain protein ipf1/pdx1 ( insulin promoter factor 1/pancreatic and duodenal homeobox 1 ) , an essential regulator of early pancreatic development . when pancreatic explants were exposed to shh , ipf1/pdx1 induced constitutive shh overexpression in the pancreatic bud , thus promoting pancreatic mesoderm differentiation into smooth muscle and interstitial cajal cells , which are typical of the developing intestine rather than the pancreatic mesenchyme and spleen . inhibition of hedgehog signaling with the specific inhibitor cyclopamine would induce a variety of changes in the pancreas , for example , gastric explants expressing endocrine pancreatic markers such as insulin and glucagon , an increase in islet cell number and size and the high level expression of ipf1 . the experiment conducted by diiorio in zebrafish clarified that gene mutation of shh and the relative signaling pathway had the specific dependence with endocrine function . these mutants confirmed that during gastrulation , the transient shh signaling induced the pancreatic endoderm to differentiate into the islet tissue subsequently . in the later development , a second hedgehog - dependent activity appeared which was similar to role of shh in the foregut endoderm assisting the localization of pancreas . the biological function of hedgehog signaling in the pancreatic epithelium remains controversial , while activation or inhibition of hedgehog signaling pathway only has slight effect on epithelial development . it suggests that hedgehog pathway might mainly focus in pancreatic mesenchyme . during embryo development , beta - cell was transiently delayed due to the pdx1-driven loss of smo that lau and hebrok discovered in pancreatic epithelial progenitor cells of pdx1/smo mice . after given birth , beta - cell numbers would restore ; however they were dysfunctional . in this case , the phenomenon ( reduced insulin secretion , slight insulin - dependent diabetes , and enhanced insulin sensitivity ) occurred . the primary cilium that was structurally required as signaling downstream of smo had close relationship with the mild responsiveness to the activation of hedgehog signaling in pancreatic epithelial cells . overactivation of hedgehog pathway in mature epithelial cell specifically with resection of primary cilium caused the following results that pancreas tissue dedifferentiated , markers of progenitor cells reexpressed , and endocrine area decreased . pscs are myofibroblast - like cells that can switch between quiescent and activated phenotypes , such as hepatic stellate cells , and reside in exocrine areas of the pancreas . quiescent hedgehog signaling is vitally important for proper differentiation and development of the pancreas . when pathogens lead to pancreatic fibrosis or carcinoma , the sensitive signaling of the hedgehog pathway is easily activated . activated pscs will migrate to the injured location and participate in tissue repair activities by secreting ecm components . research by shinozaki et al revealed in an in vitro study that exogenous ihh protein could enhance the migrational ability of pscs by increasing membrane type-1 matrix metalloproteinase on the plasma membrane , while gli1 negatively regulated ihh stimulated pscs migration by inhibiting active mt1-mmp . however , the data from sicklick et al were not consistent with this study . this could be because in different organs , hedgehog signaling may function differently . myofibroblasts were detected in capan-2 tumors , thus supporting the hypothesis that these represented pancreatic stellate cells within the tumor and proliferated in response to shh stimulation . jennifer and colleagues stimulated pscs with recombinant shh for 24  hours at 1 and 10  g / ml , and observed an increase in the mesenchymal markers sma , vimentin and desmin and a decrease in the epithelial marker ck19 . this was the first study to show that shh induces the differentiation of pscs into myofibroblasts . however , in the stroma , tumor cells overexpressing shh led to the consequences that activated the hedgehog signaling pathway in pscs instead of themselves . activated pscs were crucial in enhancing neural invasion of pancreatic cancer along axons and nerve growth of cancer cell colonies that assisted pancreatic cancer cell migration . in vivo pancreatic cancer model , paracrine shh activated the pscs and finally resulted in the tumor cell invasion of the trunk , sciatic nerve dysfunction , and the growth and metastasis of the orthotopic xenograft tumor , while the inhibitor cyclopamine could impede these pathological changes . expression of hedgehog signaling pathway components revealed that smo and gli were primarily observed in stromal - derived pscs , whereas the ligands shh and ihh were limited to pancreatic tumor cells . the development of pdac tumors was not affected when smo was genetically ablated in the pancreatic epithelium of pdac - susceptible mice . treatment of pscs with shh and ihh activated the hedgehog signaling pathway ( the upregulation of gli mrna supports this finding ) , and stimulated the proliferation of pscs . hedgehog signaling ligands secreted from neighboring cancer cells stimulated pscs to produce soluble factors that then fed back on cancer cells to promote their activity . when the pancreas suffers injuries , pscs undergo morphological and functional changes to become activated to express -smooth muscle actin . they will proliferate , migrate , and secrete ecm proteins , thus leading to pancreatic fibrosis . hedgehog signaling induces progressive pancreatic fibrosis intermingled with proliferating ductal structures that are accompanied by the destruction of acinar structures . paracrine hedgehog signaling activates myofibroblasts and leads to their proliferation , and it shows the restricted expression of hedgehog signaling downstream components including ptch , smo , and gli1/2 in hedgehog - responsive cells , while hedgehog signaling ligands induce matrix metalloproteinases ( mmps ) in all hedgehog - responsive cells , as shown in figure 2 . the hedgehog signaling pathway can be expressed in the embryonic mouse pancreas and is also observed in the adult mouse pancreas . because ihh expression is elevated in the pancreas of patients with chronic pancreatitis , ihh is thought to participate in chronic pancreatic injury , especially pancreatic fibrosis . islet cells of tissues undergoing chronic pancreatitis exhibit an abnormal localization pattern for ihh . as a signal transducer , smo is upregulated in cancer - associated stromal fibroblasts , and the expressed smo could transduce the shh to activate gli1 expression . however , ihh does not directly activate pscs nor stimulate psc proliferation ; in other words , the parameter of pscs transformation ( expression of collagen-1 or alpha - smooth muscle actin ) stays unresponsive to ihh . ihh has a powerful effect on psc migration not only in chemotactic manner but also in chemokinetic way . when target cells get stimulated and need to migrate , ihh raises the level of membrane - type 1 matrix metalloproteinase ( mt1-mmp ) and shifts them onto the plasma membrane from original location . according to the result from satoshi shinazaki and his colleagues , we could conclude that ihh promote pscs migration via gli1-dependent signaling pathway . they applied adenovirus to cause gli1 overexpression and rna interference technique to reduce gli1 expression . the results that overexpression of gli1 nevertheless blocks mt1-mmp localization on the plasma membrane and pscs migration while rna interference - mediated knockdown of gli1 augments pscs migration indicated that there was a negative feedback loop in the relationship between ihh and gli1 . during in vitro wound healing , shh increases the ability of myofibroblasts to migrate into a wounded area as a monolayer . shh significantly increases the ability of myofibroblasts to recover from the wound and promotes the migration and invasion of human pancreatic myofibroblasts in vitro . there are 3 hedgehog signaling coreceptors , gas1 , boc , and cdon , which are cell - surface - associated proteins that act as pathway activators and are expressed in cancer - associated fibroblasts . the deletion of 2 coreceptors ( gas1 and boc ) in fibroblasts leads to a reduction in hedgehog signaling responsiveness . additionally , these fibroblasts promote greater tumor growth and tumor - associated vascularity . the deletion of all 3 coreceptors ( gas1 , boc , and cdon ) results in the almost complete abrogation of hedgehog signaling and a corresponding failure to promote tumorigenesis and angiogenesis . in general , the study by mathew et al identified a role for hedgehog dosage in the promotion of pancreatic cancer and interpreted the clinical failure of blocking the hedgehog signaling pathway as a therapeutic approach in pancreatic cancer . referring to tumorigenesis , hedegehog signaling has the nonnegligible relationship . the role of hedgehog signaling relating to carcinoma once loss - of - function mutations in ptch or mutations in smo , it leads to sustained activation of hedgehog pathway . overexpression of gli1 and hedgehog proteins is associated with a variety of cancers and implicated in the onset of pancreatic ductal neoplasia and the maintenance of advanced cancers . not as we expected , in pancreatic cancer , ligand - dependent activation of hedgehog signaling instead of genomic mutation was reported and overexpression of shh was considered adequate to trigger the initiation of pancreatic cancer . as the vital effect of hedgehog signaling on pancreatic cancer , many other factors may interact with it to influence the development of the malignant disease . perineural invasion is a common pathologic feature in pancreatic cancer by which cancer cells invade the peripheral nerves and are disseminated . several studies showed that stromal pscs may play a regulatory role in the interaction between cancer cells and nerves . specific cell types and tissues that release bioactive shh from pancreatic cancer cells can produce heparan sulfate ( hs ) . according to in vivo knockdown and in vitro cell culture studies , glypican hs proteoglycans release bioactive shh morphogens from the surface of transfected bosc23 cells through hs chains in a cell autonomous manner . hs specifically modifies shh processing on the cell surface , and purified glycosaminoglycans enhance the proteolytic removal of n- and c - terminal shh peptides under cell - free conditions . the overexpression of shh in tumor cells activates the hedgehog signaling pathway in pscs in the stroma rather than activating tumor cells directly to augment the output of mmp2 , mmp9 , and ngf . these activated pscs and associated molecules promote pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies . coimplantation of pscs activated by paracrine shh induces tumor cell invasion of the trunk , stimulates nerve dysfunction , and promotes orthotropic xenograft tumor growth , metastasis , and perineural invasion in vivo . in recent years , several experiments proved that in both vivo and vitro , the hippo pathway exerted regulator ability of cell density . when the cell density exceeded the threshold , the hippo cascade would be turned on and lead to the activation of large tumor suppressor ( lats ) kinases , finally causing the phosphorylation of yes - associated protein and its paralog taz ( yap / taz ) . in cell culture , the activity of hedgehog signaling is dependent on cell - to - cell contact . when the cell intimately contacted with each other or the intensity reached high enough , hedgehog pathway activity would be regulated to higher level . adenovirus - mediated yap overexpression nevertheless prevents hedgehog signaling while the knockdown of yap expression rna interference augments hedgehog / gli activity . however , hedgehog signaling enhances the post - transcription of yap to promote its activity . however , in human and mouse pancreatic cancers , low hedgehog signaling pathway activity accompanies strong nuclear yap immunoreactivity . but tumor could utilize both ocogenic pathway simultaneously on the condition there exist protease - activated receptors ( pars ) which has the potential ability to override the hippo / hedgehog pathway . as a converse effect of yap , brd4 , a regulator of epigenetic proteins that can activate shh members in a ligand - independent manner in pdac cells , suppression of brd4 damaged pdac cell viability and proliferation and negatively influenced the tumor growth rate . combination treatment of gemcitabine and brd4 silencing has a synergistic effect on chemotherapeutic efficacy and significantly promoted apoptosis in the panc-1 and miapaca-2 cell lines . its activity in pancreatic fibroblasts leads to the expression of il-6 , an inflammatory cytokine that activates stat3 in pancreatic cancer cells . improper stromal remodeling and persistence of the inflammatory infiltrate in pancreatic tumorigenesis is a consequence of the deletion of gli1 , while partial loss of gli1 affects fibrogenesis and the recruitment of immune cells . il-6 , mil-8 , mcp-1 , and m - csf ( csf1 ) , as a subset of cytokines , potentially direct gli1 target genes to mediate this phenomenon . micrornas ( mirnas ) are a class of small noncoding rnas that play important roles in carcinogenesis . smo is a direct target of mir-125b , mir-193b , mir-324 - 5p mir-326 , and mir-338 - 3p . downregulation of mir-125b , mir-193b , mir-324 - 5p , mir-326 , and mir-338 - 3p in human cancers derepress smo and promote tumor proliferation and invasion through aberrant hedgehog signaling . mir-324 - 5p downregulation is caused by deletion of the mir-324 - 5p gene in a high percentage of mbs as a consequence of the loss of chromosome 17p . deletion of chromosome 17p leads to the loss of certain genes , thus contributing to hedgehog induced tumorigenesis . the levels of tumor suppressor mir - let7b , which targets several genes ( k - ras , muc4 , ncoa3 , hmga2 , tgfr1 , and stat3 phosphorylation ) involved in pdac pathogenesis , are down - regulated . kumar et al found that the combination therapy of mir - let7b and gdc-0449 effectively inhibited tumor growth when injected to athymic nude mice bearing ectopic tumors generated using mia paca-2 cells compared with micelles carrying gdc-0449 or mir - let7b alone . gain - of - function and loss - of - function experiments show that a pro - oncogenic function of mir-212 exists in pdac . this suggests that mir-212 could facilitate pdac progression and metastasis by targeting ptch1 , implicating a novel mechanism for the progression of pdac . mirna is not the only relevant nucleic acid , as some discoveries in dna have been made as well . twenty - five members have been reported to belong to the s100 family in humans . twenty - one of them are known as the epidermal differentiation complex ( edc ) involved in epithelial - derived cell differentiation , which are coded by genes clustered at chromosome locus 1q21 . s100a2 , s100a4 , s100a6 , s100a11 , and s100a14 from the s100 gene family are found to be significantly down - regulated due to gli1 knockdown . s100a4 and vimentin genes are up - regulated significantly by increased shh / gli1 expression , while e - cadherin is significantly reduced at the same time . migration of pancreatic cancer cells is increased significantly with dose - dependent increases in gli1 expression . silencing of s100a4 significantly reversed the response of pancreatic cancer cells induced by l - shh transduction . hedgehog signaling pathway inhibitors accelerate rather than delay the progression of oncogenic kras - driven disease . this finding was substantiated in 3 distinct genetically engineered mouse models used to study the effects of genetic or pharmacologic inhibition of the hedgehog signaling pathway activity . the balance between epithelial and stromal elements is notably influenced by pharmacologic inhibition of hedgehog , thus suppressing stromal desmoplasia . in contrast , the activation of this pathway using a small molecule agonist causes stromal hyperplasia and reduces epithelial proliferation . the stromal response to hedgehog signaling is protective against pdac , and the pharmacologic activation of the pathway can slow tumorigenesis . thus , it may offer an explanation for the failure of hedgehog inhibitors in clinical trials and may offer new insights into novel therapeutic interventions . pancreatic cancer is a highly aggressive carcinoma with an ultrahigh case fatality rate due to poor diagnosis and the lack of proper therapies . considering the unique role hedgehog signaling plays in pancreatic cancer , many strategies are focused on utilizing this pathway as a diagnostic tool for pancreatic carcinoma . clinical studies showed that the levels of shh in human blood were lower in patients with pancreatitis and pancreatic cancer compared with healthy individuals . reduced plasma levels of hedgehog were found in pancreatic cancer patients , but they were insufficient alone to predict pancreatic cancer . the epithelial - to - mesenchymal transition ( emt ) is a common phenomenon in various cancers . hedgehog signaling controls emt , enhances cell proliferation in an mapk- and pi3-kinase - dependent manner , decreases apoptosis through the regulation of bcl-2 and bcl - x , and induces the proliferation of cancer stem cells . patients with gli1 and mmp9 coexpression have poor overall survival . silencing of gli1 alone without external stimulus had no effect on emt , but inhibited tgf-1 and egf - induced emt . it performs a protumor role in the aggressive invasion of pscs by promoting tgf-1 and egf - induced emt . shh and the clinical outcome in esophageal cancer demonstrated that gli1 was a strong and independent prognostic factor for a poor outcome . furthermore , gli1 and shh are independent of the major clinical features known to influence prognosis and could serve as an adjunct to current staging systems . importantly , no interaction was observed among gli1 , shh , and adjuvant treatment activity , ensuring that this confounding factor will not affect the relationship between gli1 and shh . in resected pancreatic ductal adenocarcinoma ( pdac ) , gli1 expression is essential for pdac cell survival by facilitating the migration and invasion of cells by promoting emt . lower expression of gli1 or shh in tumor cells contributed to longer disease free survival ( dfs ) and overall survival ( os ) times . the combination of shh and gli1 levels is thought to be the most significant predictor for overall survival of os . previous studies demonstrated that activation of both the nf-b and hedgehog signaling pathways played prominent roles in the initiation and progression of pancreatic cancer by acting not only in the cancer cell itself ( autocrine ) but also in stromal cells ( paracrine ) , while the expression of either nuclear or cytoplasmic nf-b was a poor prognostic factor for pancreatic cancer . nuclear expression of either gli1 or nf-b ( rela / p65 ) is associated with poor overall survival in pancreatic cancer patients , and nuclear expression of nf-b was the only significant prognostic factor according to multivariate analysis . in conclusion , nf-b expression , mmp9 expression , and gli1 expression may have prognostic significance in advanced pancreatic cancer and could be used as biomarkers to guide therapies for pancreatic adenocarcinoma in the future . most pancreatic diseases have poor outcomes such as pancreatic fibrosis and carcinoma . as a physiologic and pathologic process , fibrosis in the pancreas has a firm relationship with pancreatic cancer . due to considerable roles in 2 lethal pathologies , the hedgehog signaling pathway has become a focus of new treatment strategies . with an increased understanding of this pathway in relevant fields , more useful and accurate methods to detect and treat these terrible diseases hedgehog signaling transduction and its regulatory factors are the basis of this promising field . currently , the mechanism underlying the failures in the clinical application of this pathway is being discovered , and these findings will go a long way toward assisting doctors in the development of new therapies .
abstractthe hedgehog signaling pathway was first discovered in the 1980s . it is a stem cell - related pathway that plays a crucial role in embryonic development , tissue regeneration , and organogenesis . 
 aberrant activation of hedgehog signaling leads to pathological consequences , including a variety of human tumors such as pancreatic cancer . 
 multiple lines of evidence indicate that blockade of this pathway with several small - molecule inhibitors can inhibit the development of pancreatic neoplasm . 
 in addition , activated hedgehog signaling has been reported to be involved in fibrogenesis in many tissues , including the pancreas . therefore , new therapeutic targets based on hedgehog signaling have attracted a great deal of attention to alleviate pancreatic diseases . in this review , we briefly discuss the recent advances in hedgehog signaling in pancreatic fibrogenesis and carcinogenesis and highlight new insights on their potential relationship with respect to the development of novel targeted therapies .