The past decade has seen significant advances in our understanding of Hedgehog (HH) signaling pathway in various biological events. HH signaling pathway exerts its biological effects through a complex signaling cascade involved with primary cilium. HH signaling pathway has important functions in embryonic development and tissue homeostasis. It plays a central role in the regulation of the proliferation and differentiation of adult stem cells. Importantly, it has become increasingly clear that HH signaling pathway is associated with increased cancer prevalence, malignant progression, poor prognosis and even increased mortality. Understanding the integrative nature of HH signaling pathway has opened up the potential for new therapeutic targets for cancer. A variety of drugs have been developed, including small molecule inhibitors, natural compounds, and long non-coding RNA (LncRNA), some of which are approved for clinical use. This review outlines recent discoveries of HH signaling in tissue homeostasis and cancer and discusses how these advances are paving the way for the development of new biologically based therapies for cancer. Furthermore, we address status quo and limitations of targeted therapies of HH signaling pathway. Insights from this review will help readers understand the function of HH signaling in homeostasis and cancer, as well as opportunities and challenges of therapeutic targets for cancer. Some targeted tumor therapies have entered clinical trials as the mechanism of HH signaling pathway is further explored.7 The current review aims to provide a comprehensive background on HH signaling pathway and highlight the recent advances of its role in homeostasis and tumorigenesis. It will also shed light on the available means and potential challenges of targeted therapies for HH signaling pathway-dependent cancer.HH signaling pathway was originally identified for its role in patterning the Drosophila embryo development.8 Signal reception is achieved by conserved receptors on the cell membrane, including the 12-pass transmembrane protein Patched (Ptch) and the 7-pass transmembrane protein Smoothened (Smo). In the absence of hedgehog ligand, Ptch inhibits the activity of Smo causing pathway inhibition. When the hedgehog ligand binds to Ptch, the inhibition of Smo is lifted, thus continuing to activate downstream signaling.9 In addition to this, the activation of hedgehog signaling requires co-receptors, which can be involved in the inactivation of Ptch.The initiation of Hedgehog signaling is based on the processing of Hedgehog ligands in secretory cells, where a dual lipid-modified amino-terminal  polypeptide of the autocatalytically cleaved precursor protein is formed.10 Gli proteins include an amino-terminal transcriptional repression domain, a zinc finger DNA binding domain and a carboxy-terminal transcriptional activation domain. When transcription is not activated, Gli is processed by protein hydrolysis into the form of a repressor with a truncated carboxy-terminal activation domain, which inhibits the transcription of the target genes.11 In addition, Suppressor of fused (Sufu), a negative regulator of Gli, is essential for pathway activity in mammals.12In mammals, hedgehog signaling activation elicits a transcriptional response in the nucleus of the recipient cell that is mediated by glioma-associated oncogene (Gli) transcription factors.13 The primary cilium consists of ciliary membrane, axoneme and basal body. The presence of intraflagellar transport (Ift) proteins on the axoneme mediates the transport of signaling proteins, and this transport mode can regulate the concentration of the corresponding proteins within the cilium.14 In addition, primary cilia form compartments that are separated from the rest of the cytoplasm of the cell and thus provide a unique intracellular environment for pathway components.15A particular aspect of mammalian hedgehog signaling is the transport of signaling molecules at all levels in the primary cilium.16In this part, we will briefly introduce the signaling process to understand the mechanism of HH signaling pathway in tissue homeostasis and tumorigenesis. A large body of researches have reported the core components of HH signaling pathway. This cascade includes extracellular Hedgehog ligands; cell surface receptors and co-receptors; Gli family transcription factors (Gli1-Gli3) containing zinc-lipid structures and numerous intracellular and extracellular co-regulators.17 Three soluble extracellular Hedgehog ligands have been identified in vertebrates, including Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh), which have distinct distributions in different developmental stages and tissues.19Hedgehog ligands are paracrine signaling factors that can mediate communication between cells up to hundreds of micrometers away.21 Moreover, Shh directs the growth of post commissural axons along the longitudinal axis of the spinal cord, acting as a major mediator during central nervous system development.23 Collectively, Shh plays a vital role in the developmental regulation of various tissues such as skull, adrenal cortex, hair follicles and sweat glands.27Shh-triggered signaling is well documented for its critical role during embryonic and postnatal developmental processes. For example, Shh forms a ventral and dorsal concentration gradient in the neural tube, thereby directing neural progenitor cell fate in time and space as well as acting physiologically as a morphogenetic agent.8 The secretion of activated Shh serves a crucial role in regard to its biological function. Shh was first cleaved into a 19 kDa Shh amino-terminal polypeptide (Shh-N) and Shh carboxy-terminal polypeptide (Shh-C). Then, Shh binds cholesterol to the carboxyl terminus of Shh-N through a protein hydrolytic cleavage process, whereas, Shh-C exits the endoplasmic reticulum and enters the proteasome to be degraded, ending its mission.29 In general, palmitoylation occurs after cholesterol modification, further increasing Shh activity.30 Palmitoylation of Shh is mediated by HH acyltransferase (Hhat), which transfers palmitic acid to the amino-terminal cysteine residue of Shh-N.32 The activated form of Shh is a dual lipidation of cholesterol and palmitic acid by Shh-N  is localized on the surface of secretory cell lipid membranes, where it interacts with Shh-N and recruits the lipoprotein lipophorin, resulting in the loading of Shh-N on lipoprotein for transport.39 In addition to the ability of secretory cells to positively regulate Shh secretion, Ptch1, which receives Shh receptors on the cell surface, is able to participate in the negative feedback regulation of ligands, limiting the range of Shh signal waves.40 In conclusion, Shh is able to assemble into suitable carriers for transport, and other possible mechanisms that help Shh diffusion deserve further investigation.The mechanism by which the diffusion of activated Shh from secretory cells occurs is more complex Fig. . Dispatc42 Expressed by Sertoli cells, Dhh directly mediates the proliferation and differentiation of spermatogonia through the regulation of downstream signaling pathways. Dhh-deficient animals exhibit abnormal testicular mesenchymal cell differentiation and defective peritubular cell morphology, resulting in sterility in male mice.43 The function of Ihh is mostly related to chondrocyte differentiation in the skeleton.44 It regulates the differentiation of growth plate chondrocytes through a negative feedback mechanism of parathyroid hormone-related proteins, and also directly controls chondrocyte proliferation and osteoblast function.45Dhh and Ihh act more specifically than Shh and show pronounced tissue specificity. Dhh is a key regulator of gonadal tissue development, especially in ovarian granulosa cells and spermatogenesis.46 This N-terminal structural domain is then modified by N-terminal palmitoylation as well as the cholesterol addition of C-terminal amino acids, resulting in the formation of the mature proteins Dhh-N and Ihh-N.47 All three HH ligands share high sequence homology, similar overall fold and are capable of binding to and activating the same signaling pathway receptors. They mainly differ in their N-terminal amino acid sequence; however, their N-terminal sequence similarity may also reach 76–91%.48The activation and secretion of Dhh and Ihh are similar to that of Shh. Dhh and Ihh also undergo autocatalytic cleavage to form Dhh-N and Ihh-N, in which the C-terminal structural domain is removed.49 The potential roles of Dhh and Ihh in varies organs requires further investigation.Over the years, it has also become increasingly clear that Dhh and Ihh have multiple functions in other tissues. For instance, full-length Dhh plays a critical role in regulating vascular endothelial integrity.50 The structure of Ptch1 includes the transmembrane structural domain (TMD), which comprises a sterol-sensing structural domain (SSD) and two extracellular structural domains (ECD1 and ECD2)  Fig. .51 Variolex Fig. .53 Howev54 Ptch2 may be able to bind HH proteins in different cellular environments in order to regulate the subsequent signaling pathway.55While the role of Ptch2 is inadequately understood, it compensates the response to Shh in the absence of Ptch1.57 The TMD and extracellular loops form a ligand-binding region.58 CRD at the extracellular amino terminus mediates Smo dimerization. In addition, intracellularly, the carboxy-terminal tail mediates the conformational change of the Smo dimer. In the absence of ligand, the carboxyl terminus is in a closed conformation, obscuring the kinase binding site; in the presence of ligand, the carboxyl terminus ser/thr cluster is highly phosphorylated, forming an open conformation.61In-depth structural and functional studies have revealed the role of the two variable ligand binding sites, seven-pass α-helical transmembrane bundle (TMD) and extracellular cysteine-rich domain (CRD) of Smo, a G protein-coupled receptor, in regulating Smo activity Fig. .56,57 Th63 The inactive Smo is located in the cytoplasm. However, when HH binds to Ptch, the restrictive effect of Ptch on Smo is lifted to initiate downstream signaling pathways.65 Ptch is transported to lysosomes in the cytoplasm for degradation. Smo activation requires two mechanisms: first, Smo needs to be translocated to primary cilia, a microtubule-based organelle  and growth arrest-specific 1 (Gas1) have been identified as co-receptors regulating HH signaling pathway activity. The specific heterogeneous complexes formed by their interaction with Ptch1/2 receptors, respectively, can mediate different kinetic Smo depression programs through distinct ligand reception patterns.74 Through protein hydrolysis, they act as bifunctional transcription factors, changing from the full-length transcriptional activator form (Glia) to the carboxy-terminal truncated transcriptional repressor form (Glir).11 GLI-similar (Glis) transcription factors, a subfamily highly similar to Glis, can also undergo nuclear translocation by post-translational modifications in the cytoplasm, and their potential interconnection with Gli1-3 enriches the Gli network.77Upon excitation of HH signaling pathway, Gli transcription factors are activated thereby regulating target gene expression. The vertebrate homologs of this Drosophila Cubitus interruptus transcription factors include Gli1, Gli2, and Gli3 proteins. Gli1 is exclusively a transcriptional activator, whereas Gli2 and Gli3 have dual activity. Although both contain highly similar zinc finger structural domains that can bind to DNA, Gli3 often acts as a transcriptional repressor because the process of protein hydrolysis is most pronounced at Gli3, while Gli2 acts primarily as a transcriptional activator.79 In cells, Sufu is phosphorylated by Protein kinase A (PKA) to form a stable phosphorylated form of Sufu, which affects its interaction with Gli.80 Sufu and Gli form a complex in the cytoplasm after entering the primary cilia  and positive (Gli1) regulation of HH signaling with feedback loop mechanisms. Furthermore, in tissue-specific stem cells, HH signaling induces Bmi1, which acts as a transcriptional repressor that regulates the proliferative capacity of cells.85 Numerous target genes in this pathway are cell cycle regulators, such as Myc, CyclinD, and CyclinE, as well as development-related regulators, such as Fgf4 and Hhip.89 In the developing neural tube, this pathway regulates the Nkx6.1, Olig2, Nkx2.2, and FoxA2 genes, conferring ventral neural fates and has Gli-binding sites in the regulatory regions.90 In addition, HH signaling pathway promotes the expression of certain genes, including Snzi1, Zeb1, Twist2 and Foxc2, which enhance epithelial mesenchymal transition.91 Other target genes include the apoptosis regulator Bcl2, Vegfa, Abcg2, Mycn, Foxm1, Pax6, Pax7, Pax9 and Jag1, as well as members of the Wnt signaling pathway.98 In summary, HH signaling pathway can regulate the corresponding target gene transcription in different functional states and cell types.HH signaling pathway plays an essential role in cell proliferation and differentiation, tissue development, homeostasis and EMT.99 This section will briefly describe the structural features of primary cilia and the way in which the key components of HH signaling pathway act in primary cilia.Unlike other signaling pathways, the normal maintenance of the vertebrate canonical HH signaling pathway is highly dependent on primary cilia. Primary cilia recruit extracellular signaling components that link the extracellular environment, where signals are complex and diverse, to the intracellular environment.100 The axoneme of primary cilia includes 9 microtubule doublets in the outer ring, while lacking the central 2 microtubule singlets (known as 9 + 0 axoneme). Primary cilia are anchored to the cell by basal body, which is specialized from the mother centriole and contains a number of proteins for ciliogenesis and assembly.102 Between the cilia and basal body is the ciliary transition zone, which regulates protein transport within the cytoplasm and cilia.103The primary cilium is a specialized structure located on the cell surface.14 Ift is driven by two molecular motors, involving cytoplasmic dynein2, which mediates transport from tip to base, and the heterotrimeric kinesin-2 complex comprised of Kif3a, Kif3b, and Kap3, which is responsible for transport from base to tip.105 Mutations in the gene encoding the heavy chain of cytoplasmic dynein2 cause axoneme swelling because Ift-related proteins are trapped in cilia due to disruption of retrograde transport.14 Gli cooperates with Kif3a and Kap3, where the interaction of Kap3 restricts Gli transcriptional activity.106 Ift-B complex protein and Ift-A complex protein together constitute Ift trains that bridge kinesin motors with HH signaling molecules.107 Ift trains slide back and forth along the axoneme and are responsible for cilia structure assembly and balancing. Periodic binding of Ift-A, Ift-B and dynein2 can balance the protein entry and exit of cilia, effectively preventing the accumulation of material.109 Huang et al. showed that wimple mutants disrupting Ift172, an Ift-B protein, lacked the normal primary cilia structure, which resulted in the inability of the receiving cells to respond to Shh.110The transport of protein molecules within primary cilia to the cytoplasm is achieved through the Ift mechanism.111 Disruption of most primary ciliary proteins decreases signaling activity, but some proteins are altered in different ways that enhance pathway activity; thus, changes in ciliary structure have a bidirectional effect on the HH signaling cascade response, with subsequent effects on cell fate.112Changes in the structure and function of primary ciliary protein fractions can have an impact on HH signaling pathway. Defective RNAi of Kinesin Kif14 disrupts the function of distal appendage proteins Sclt1 and Fbf1, which results in a defective basal body that fails to activate HH signaling pathway.114 In the absence of Hedgehog ligand, Ptch induces a closed conformation of Smo by controlling the TMD binding of small lipid Smo ligands to Smo, allowing Smo to remain in the cytoplasm  and G protein-coupled receptor kinase 2 (GRK2) phosphorylation , cAMP increases and activates PKA, which phosphorylates both proteins of the Sufu/Gli complex.125 Gli is further phosphorylated by CK1 and Glycogen Synthase Kinase-3 (GSK3β) and forms Glir by proteolytic cleavage  and osteoblast markers. Tissue engineering experiments utilizing Ihh-MSCs-scaffold complex has shown increased bone repair capacity.145 Shh is a potential signaling molecule that regulates osteoblast differentiation. Armstrong et al. have shown that Shh in zebrafish can upregulate the expression of Sp7 in osteoblast lineage cells and increase the proliferation of osteoblasts.146HH signaling pathway is one of the major signaling pathways regulating osteogenesis and post-embryonic long bone homeostasis with postnatal expression in the mesenchyme.148 Zhao et al. revealed that cells harboring MSCs characteristics were Gli1+. These MSCs play a significant role in regulating calvarial bone formation and injury repair.147 Using diphtheria toxin to disentangle Gli1+ cells led to premature fusion within the cranial sutures, resulting in craniosynostosis, indicating the vital role of HH signaling pathway in controlling the suture homeostasis and calvarial bone patterning and repair.149 Moreover, the interplay between BMP and Ihh signaling has been identified in maintaining suture homeostasis via interaction among MSCs, osteoprogenitors, and osteoclasts during calvarial bone formation and repair.150The calvarial bone has become an emerging model for studying the function of HH signaling pathway in bone homeostasis. Regarded as the primary niches for osteogenesis, craniofacial sutures contain MSCs.151 Myosatellite cells normally maintain a quiescent condition and give rise to myogenic cells and reform the myofibers of the muscle in response to injury.152 Study showed that muscle injury could stimulate Shh expression. When Shh signaling was inhibited, the expression of Myf5 and MyoD was impaired, accompanied by reduced number of activated myosatocytes.153 Similarly, Koleva et al. showed that Shh could promote the proliferation of myosatellite cells and myotube fusion.154 Subsequently, Elia et al. found that Shh can promote the differentiation of chicken primary myoblasts, and this effect can be inhibited by cyclopamine, an inhibitor of Shh signaling.155 It has also been reported that Shh promotes the expression of Pax7, Myf5, MyoD, MyoG, and MyHC in mouse myoblasts, and the promotion of these genes by Igf-1 depends on HH signaling pathway.157 Further study by Voronova et al. showed that Gli2 formed a protein complex with MyoD and Mef2c to enhance the transcription factor activity of MyoD.158 MyoD regulates the activity of HH signaling pathway, which allows Gli2, Mef2c, and MyoD to form a reciprocally regulated recycling network. Devakanmalai et al. reported that HH signal affects muscle cell differentiation by activating MyoD and Myf5, and then regulating Mef2c. These studies indicate that HH signaling pathway is essential for postnatal muscle homeostasis.159Skeletal muscle has the ability to repair damage due to the presence of muscle stem cells, namely, myosatellite cells.+ cells were progenitors of vascular smooth muscle cell and expressed CD34, Sca1, and Pdgfrβ, possessing tri-lineage differentiation capacity towards osteoblasts, adipocytes, and chondrocytes.160HH signaling pathway may also participate in smooth muscle regeneration and differentiation. Kramann et al. found that adventitial MSC‐like Gli1162 Mammalian epidermal metabolism is maintained by the continuous proliferation of epidermal stem cells.163 Adolphe C et al. found that Ihh overexpression did not result in any significant epidermal morphogenetic phenotype, while overexpression of Dhh was indistinguishable from Shh. The phenotypes were resulted from dysregulation of stem cell activity, including hyperplasia of epidermal progenitors and almost complete loss of epidermal tissue renewal capacity, indicating that HH activity is the vital factor to maintain homeostasis of epidermal stem cells.164 Zhou et al. showed that Shh, its receptors Ptch1, Smo and its downstream transcription factor Gli1 were detected in the basal layer of fetal epidermis and in newly sorted human putative epidermal stem cells (HPESCs).165 HPESCs treated with medium containing Shh-N exhibited enhanced cell proliferation. In contrast, cyclopamine inhibits Shh and thus prevents proliferation. Similarly, the mitogenic effect of epidermal growth factor on HPESCs can be eliminated by cyclopamine. In addition, the expression of Bmp-4, a potential downstream effector of Shh signaling, can increase HPESCs proliferation.165 Shh signaling in mammalian skin controls hair follicle epithelial cell growth and morphogenesis by regulating Gli transcription factors.166 Shh expression and the ability of skin cells to respond to Shh signaling pathways are spatially and temporally regulated during the hair cycle.167 Knockout of Shh-dependent Sox9 in the skin results in the appearance of external hair, with severe proliferation defects and difficulty in forming a stem cell microenvironment.168 Additional studies have shown that the demand for substrate formation by β-catenin occurs downstream of Tabby/Downless and upstream of Bmp and Shh.169During the hair cycle, Shh signaling pathway is essential for maintaining hair follicle stem cell populations and regulating the development of hair follicles and sebaceous glands, meanwhile regulating epithelial-mesenchymal interactions.170Villin-Cre;Ihhflox/flox mutant mice show a significantly reduced number of villi and reduced cell proliferation in the stem cell compartment, suggesting that Ihh is essential for ISC regeneration and differentiation.171 Specifically, deletion of Ihh in intestinal epithelial is accompanied by an increase in epithelial Wnt signaling pathway, while the activation of the Wnt pathway is a common cause of intestinal tumorigenesis.173 HH might have a tolerogenic influence as a regulator of inflammation.174 Lees et al. showed the reduced expression of HH signaling pathway in individuals with inflammatory bowel disease and ulcerative colitis.175 Dop et al. conditionally deleted Ihh in adult mice and found an inflammatory response accompanied crypt changes, resulting in influx of macrophages and fibroblasts into the villus core.176 Recent studies suggest that more than one target cell population  is relevant to HH-modulated inflammation.178Intestinal epithelial homeostasis is regulated by a strictly controlled balance between intestinal stem cell (ISC) proliferation and differentiation.179 Tien et al. found proliferative expansion of the adjacent lung mesenchyme when specifically deleting Shh in the murine lung.180 Reduced expression of HH signaling pathway is initially detected during the acute phase of epithelial injury as the mesenchyme proliferates in response, whereas being upregulated to the baseline while the homeostasis is restored. However, Liu et al. demonstrated the upregulated HH signaling pathway in lung fibrosis or airway injury, supported by a proliferation in stromal Gli1+ cells in adult mice.181 Moreover, when confronted with an airway injury, Shh overexpression enhances collagen deposition and lung fibrosis.Epithelial-mesenchymal interactions coordinated by HH actively maintain homeostasis and regulates repair and regeneration in lung.182 Meanwhile, Shh signaling pathway also plays an important role in the homeostasis and regeneration of dentin, the mesenchymal compartment of incisors. Zhao et al. identified that Gli1+ MSCs were localized around arteries and the accompanying nerves, while the HH ligand that maintains the Gli1+ MSC population was secreted by nerves in the neighboring neurovascular bundle (NVB).183 In addition, Gli1+ cells were also indicated to contribute to incisor injury repair by generating reparative dentin.Both epithelial and mesenchymal compartments of the rodent incisors continue to grow and regenerate throughout the lifespan of animals and requires constant repair. It has been identified that during the growth of the mouse incisor, Shh plays a significant role in maintaining the ability of stem cells to expand the ameloblast lineage in the incisor epithelium, indicating that Shh regulates the homeostasis of mouse incisor enamel, the epithelial compartment of incisors.+ cells were distributed within the mesenchyme, mainly in the periodontal ligament near the molar apical. These cells were activated and significantly contributed to the bone remodeling when imposed on the orthodontic forces.184Unlike the rodent incisor, the rodent molar does not possess the property of continuous growth, and the majority of Gli1185 Cancers are highly burdensome diseases, which have negative physical and mental effects on patients, as well as serious economic consequences. HH signaling pathway has been documented to be responsible for tumor initiation and premalignant lesion as well as tumor progression leading to a greater tumor size and more invasive behavior.188 Tumor metastasis and the resistance to anti-cancer therapy (radio and chemo-resistance) were the ultimate challenges to fight cancer as a life-threatening disease.190 Also, cancer stem cell (CSC) with the self-renewal ability not only has a close relationship with multiple tumor properties mentioned above such as tumor initiation, development, metastasis, and tumor recurrence.191 Therefore, we mainly discussed the decisive role of HH signaling pathway in five aspects including cancer initiation, cancer progression, metastasis, resistance to anti-cancer therapy, and cancer stemness in different organs in this section  cases when compared to nonneoplastic oral mucosa. Recent study demonstrated that Gli1 was present only in the nuclei of cancer-associated fibroblasts in OSCC, indicating that HH signaling is active in the progression of OSCC.200 Furthermore, endophytic-type parenchyma of OSCC possessed a stronger expression of Shh and Ptch1 than exophytic-type parenchyma of OSCC.201Squamous cell carcinomas account for more than 90% of oral malignant tumors and often begin with precancerous lesions.203 HH signaling regulates cell proliferation in OSCC.204 Several studies have investigated the molecular mechanisms on the invasion behavior of human OSCC. Ptch1 and Gli expression were found in the microvascular cells in the invasive front of OSCC.205 Takabatake et al. reported that Ptch and CD31 double-positive blood vessels in the OSCC stroma might affect tumor angiogenesis in OSCC.201 Also, Shh overexpression is correlated with cancer stem cell markers CD133 and Sox2 in OSCC specimens.202 Shh might induce OSCC progression and invasion through prolonged half-life of activated leukocyte cell adhesion molecule (ALCAM) which is a transmembrane glycoprotein mediating cell adhesion and multiple other function of cancer cell.206 An increased MMP-9 and downregulated epithelial cadherin (E-cadherin) expression caused by Shh signaling in OSCC might be responsible for the invasiveness and metastatic potential.207HH signaling molecules are considered to be prognostic indicators in OSCC as overexpression of related proteins can be indicative to tumor size, metastatic potential, tumor recurrence and even to shorter overall survival.209 Gorlin syndrome (GS) is a multisystem genetic disease caused by gain of function mutation in HH signaling pathway and manifests as multiple OKCs and basal cell carcinomas (BCCs).210 The most common gene mutation for GS is Ptch1 while Ptch2, Smo and Sufu are also rare causative genes for GS.211 Although multiple OKCs is a major symptom of GS, even patients with sporadic OKCs were suspected with an underestimated Ptch1 mutation rate.212 Stojanov et al. demonstrated Ptch1 inactivating mutations accounts for 93% of sporadic OKCs.213 Yu et al. indicated that mutations in the intracellular loop of Ptch1 might regulate Cyclin B1 in NBCCS-associated OKCs in a manner of noncanonical HH signaling pathway.214 Ptch1 mutation leads to ligand-independent activation of Smo and subsequently upregulates HH signaling pathway target genes transcription.Odontogenic keratocysts (OKCs) accounts for 3.3–17.4% of all jaw cysts, of which neoplastic lesions often involve mandibles with a high clinical risk of recurrence.216 Grachtchouk et al. uncovered that elevated expression of HH target genes is detected in lower cell layers of cyst wall of human OKCs.217 Besides, OKCs were found to express lower levels of Sufu gene and higher level of Smo, Ptch1, Cyclin D1 and Bcl2.218 Syndromic OKCs were found to have a higher expression of HH signaling pathway protein than the sporadic OKCs.219 Higher Smo expression was considered to be a risk factor of recurrence of OKCs.220Many studies concluded that the aberrant HH signaling pathway is active in the development of OKCs.185 Two types of malignant primary brain tumors including medulloblastoma (MB) and glioma were in close relationship with HH signaling pathway.221 MB is a common aggressive malignant tumor in brain during childhood.222 There are four subtypes of MB based on research on their molecular mechanism and clinical characteristics: Wnt-MB, Shh-MB, group 3 MB and group 4 MB.223 The novel feature of Shh-MB is constitutive activation of the Shh signaling pathway.Brain tumor and other neoplasm in nervous system account for 1.6% new cases worldwide in 2020 and lead to more than 251,329 deaths.224 Researches have indicated that germline and somatic mutation of HH signaling pathway related components leads to MB and such mutation varies in different ages.225 However, Ptch1 represents the most common oncogenic mutations in Shh-MB.226 Nearly half of patients with Shh-MB harbored Ptch1 (45%) alterations while Smo (14%) and Sufu (8%) alterations were also detected.227 Mouse model with conditional deletion of Ptch1 in CGNPs caused MB formation, suggesting that CGNPs play a central role in the origin of Shh-MB.229 Astrocytes in the cerebellum perform important functions that support granule cell proliferation and migration in the physiological state while MB-associated astrocytes can secrete Shh ligand that helps maintain proliferation tumor cell in a Ptch1-independent manner.230HH signaling stimulates the proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development, which lead to MB formation.231 Quiescent cells with a therapy-resistant characteristic can serve as a reservoir for relapse. Sox2, a neural stem cell marker, is recognized as a poor prognosis marker in human Shh-MB. In a mouse model of Shh-MB, cells expressing Sox2 accounts for a proportion of less than 5% of the total tumor cells but might serve as a reservoir for relapse.232HH signaling pathway has effect on multiple targets including Mycn, Snail1, Cyclin D1, Sox2, Sox9 and further influence cancer behavior as tumor growth and tumor cell proliferation during MB tumorigenesis.233 Given to characteristics of high invasiveness and drug resistance, GBM is a neoplasm with a dismal prognosis and an effective practical therapeutic approach remains under investigation.234 Researchers have founded that HH signaling pathway is activated in human glioma cell lines, but not in cultured human astrocytes.235 HH/Gli1 pathway affects the growth of glioma and glioma stem cells are thought to be generated by the population of Gli positive neural stem cells. Researchers reported that Gli1 and Gli2 expression is associated with grades III and IV gliomas with less survival.236 The overall survival of patients with glioblastoma is decreased by upregulation of Shh based on data from the Cancer Genome Atlas glioblastoma.237Glioblastoma multiforme (GBM) is a common malignant brain tumor and account for 23% of all gliomas.238 Chang et al. enhances HH signaling pathway in GBM cells by recombinant human Shh N-terminal peptide, which increases the production of MMPs to promote cell migration and invasion through the PI3K/AKT pathway.239 Overexpression of fms related tyrosine kinase 1 (FLT1) in GBM cells was related to the invasion and migration of tumor cells through HH signaling pathway.240 Although, Shahi et al. reported a fainter expression of HH signaling pathway component in neuroblastoma (NB) than MB or glioblastoma.241 Studies also reported that HH signaling pathway determines multiple biological behavior of neuroblastoma,244 while several researchers proposed theories of a tumor-suppressive functions of HH signaling pathway in NB.245Cell migration and proliferation in human GBM cell lines are associated with expression of connexin 43, an integral membrane protein within gap junctions, upon HH signaling pathway modulation.247 Dormoy et al. reported HH signaling pathway components are expressed in human CRCC tumor samples, which participates in cell proliferation of CRCC cell lines and related tumor growth. Such reactivation of HH signaling pathway leads to regulation downstream gene transcription of Cyclin D1, PAX2, VEGF, and TGF-β.248 The mRNA levels of Shh, Smo and Gli1 were higher in CRCC tissue compared with control kidney tissue in different degrees.249 Gli3, Ptch1, Dhh, and Shh were highly expressed in in higher grade tumor as Dhh expression can be recognized as an independent predictor of CRCC survival.250 Multiple studies show antitumor potential in human RCC by targeting HH signaling pathway.252Renal cell carcinoma (RCC) is the most common renal tumor, accounting for 80–85% of all renal cancers, as clear cell renal cell carcinoma (CRCC) is responsible for the vast majority of RCC.253 HH signaling pathway may interact with hypoxia-inducible factor 2α modulating the radiosensitivity of RCC.254 Furukawa et al. suggested that Gli2 may be related to the underlying mechanism of drug resistance associated with tyrosine kinase inhibitor (TKI) inhibitors sunitinib in CRCC.255HH signaling pathway influence aggressiveness of RCC. Recombinant Shh protein enhanced cell proliferation and suppressed the expression of E-cadherin in RCC cell lines, suggesting the essential role of EMT modulating by HH signaling pathway in RCC.185 Paracrine signaling of HH signaling pathway is crucial to prostate development.234 PCa originates from prostatic epithelia and epithelial-mesenchymal interaction plays an important role in PCa progression and metastasis.257 Wilkinson et al. has shown the co-localization of Smo with primary cilia in prostatic fibroblasts and confirmed the activated HH signaling pathway in prostatic tumor microenvironment.258 Also, Shh expression level was correlated with cell proliferation in PCa. HH signaling pathway blockade by cycloplamine, a selective inhibitor of Smo, significantly reduced the proliferation of PCa cell lines.259 Enhanced expression of HH signaling pathway components was found in the cancer tissue than in the normal prostatic epithelial tissue, which was correlated with higher Gleason score and worse prognosis.261 In animal models, overexpression of Hedgehog protein persistently in mutant mice accelerated the progression of prostatic intraepithelial neoplasia that led to PCa. Hyperplastic basal cells might be the true cellular origin of primary PCa. HH signaling pathway plays important roles in transforming normal prostate basal/stem cells into PCa stem cells.262 In addition, inhibition of Shh signaling pathway showed potential for prevention of drug resistance such as zoledronic acid.263Prostate cancer (PCa) is the second most common cancer in male accounting for 14.1% new cases worldwide.265 Importantly, HH signaling pathway is influential to multiple biological behaviors of liver tumor including hepatocellular carcinoma (HCC) and Cholangiocarcinoma (CCC).HH signaling pathway is considered to play an essential role in liver organogenesis and hepatic tissue repair.185 Ptch1 was found overexpressed in HCC tissue compared with the surrounding non-neoplastic liver tissue. Moreover, increased expression of Smo and Gli1 was directly correlated to a large tumor size of HCC.267 Ptch1 and Gli1 can be potential biomarkers for the recurrence of HCC and cumulative survival of HCC patients.268 MMPs promote tumor metastasis by influencing extracellular matrix remodeling. Shh signaling pathway induces MMP-2 and MMP-9 production to promote invasiveness through FAK/AKT signaling in HCC.269 HCC’s invasive behavior was attenuated by treatment with a Smo inhibitor which partially suppressed the expression of MMPs and Gli1/2.270 In addition, target genes of HH signaling pathway such as EMT transcription factors were found overexpressed in poorly differentiated hepatoma cells in HCC. Activation of HH signaling pathway and related EMT factors might be responsible for the invasiveness and chemoresistance in poorly differentiated hepatoma cells.272The most common malignant liver tumor is HCC.273 Further experiments by Zhou et al. confirmed that Gli1/2 binds to the promoter of transporter associated with antigen processing 1 (TAP1) gene, indicating that TAP1 is one of target genes of HH signaling pathway in HCC cell lines. RNAi targeting Gli or TAP1 can alleviating drug resistance.274A specific Gli inhibitor GANT-61 significantly suppressed HH signaling to reverse sorafenib resistance in CD44-positive HCC, suggesting HH signaling pathway can influence HCC drug resistance.275 Riedlinger et al. revealed a significant activation of HH signaling pathway in CCC samples and cell proliferation of CCC cells was significantly reduced by inhibiting HH signaling pathway.276 The potential effectiveness was also confirmed in a CCC xenograft model that treatment with a combined therapy with BMS-833923 and gemcitabine inhibited tumor growth. Furthermore, HH signaling pathway components such as Gli1 and Gli2, are reliable prognostic factors for CCC.278CCC is a malignant tumor derived from biliary epithelial cells. According to different anatomical positions, it can be classified into intrahepatic, perihilar, and distal CCC, among which perihilar CCC represents about 50% of all cases.280 HH signaling pathway protein might promote the interaction between cancer cell and stromal cell and eventually further promotes cancer progression. Coculture of CCC cells with hepatic stellate cell line Lx-2 increased cancer cell migration and invasion.281 CCC cells in conventional and hypoxic conditions have been observed to promote tumor-associated macrophage polarization and TGF-β1 secretion via paracrine Shh ligands, thereby promoting CCC cell growth, metastasis and endoplasmic reticulum homeostasis via TGF-β1.282Hypoxia influences multiple tumor biological behavior. Hypoxia inducible factor-1 (HIF-1) promotes cancer stemness and invasive behavior of CCC by modulating Shh, Smo and Gli1.185 HH signaling pathway in the gastrointestinal tract relies mainly on paracrine secretion for completion. In mouse models, the overexpression of Shh ligands provided advantages in growth to tumor cells by activating HH signaling pathway in the surrounding stroma.196 Further studies have shown that Shh ligands are upregulated in CRC in order to activate the stromal HH signaling pathway, while stromal downstream genes, such as Gli1 and Hhip expression, are decreased.283 This could explain the negative results and Shh ligand overexpression that were evident in clinical trials involving vismodegib for CRC. Several studies have indicated that the downregulation of Ihh can be observed as an early event in the formation of CRC.285 Ihh is mainly expressed in the stromal tissue of the colon, and its high expression level has been described to inhibit the development of CRC.283 In a mutant mouse model, colorectal epithelial cells were shown to secrete Ihh to maintain the intestinal stromal phenotype, which was essential to adenoma development, suggesting that Ihh could influence colorectal malignancies in a variety of manners.286 Hypoxic environments can induce the binding of HIF-1α produced by tumor cells and TGF-β2 secreted by cancer-associated fibroblasts to activate Gli2 expression in CRC cells, thus leading to elevated resistance of CRC cells to chemotherapy.287Among all malignant tumors, colorectal cancer (CRC) ranks third in the world.288 Shh is aberrantly expressed in pancreatic intraepithelial neoplasia and HH signaling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas.289 High expression of Shh and Gli1 was an independent prognostic factor for worse survival of patients with pancreatic ductal adenocarcinoma (PDAC).290 Moreover, pancreatic cancer-associated fibroblasts show overexpression of Smo that lead to activation of HH signaling pathway.291Overall 5-year survival of pancreatic cancer is below 10%.293 However, Lee et al. proposed that stromal response to HH signaling pathway is protective against PDAC and that HH signaling pathway activated by SAG21k can decelerate tumorigenesis.294 Perineural invasion is an important characteristic of pancreatic cancer with an incidence of 70–100%. Cancer cells invade into peripheral nerves in pancreatic tissue and are considered to be associated with poor tumor prognosis and cancer pain.296 Moreover, overexpression of Shh activates HH signaling pathway in pancreatic stellate cells in the tumor stroma which is not only essential for tumor growth but also responsible for the nerve invasion in cancer.297HH signaling pathway influences multiple tumor cell behavior in pancreatic cancer through regulating Gli1 expression. Gli1 expression is essential for PDAC cell survival by facilitating the migration and invasion of cells by promoting EMT. S100A4 is an EMT indicator protein and might be an essential target gene mediated by Gli1 in pancreatic cancer.298 Inhibition of Shh signaling by cycloplamine induces a significant decrease in the proliferation of Non-Small-Cell-Lung-Cancer (NSCLC) cells mediated by Gli through regulation of cyclin expression. Moreover, increased Shh expression in NSCLC might be related to cancer progression mediated by cancer stroma-associated fibroblast.299Similar to many cancers, HH signaling pathway is also important for the prediction of lung cancer prognosis.301 Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are widely used as first line therapy for NSCLC. Upregulation of HH signaling pathway resulted in EGFR-TKIs resistance by EMT induction. Blockade of HH signaling increased sensitivity to EGFR-TKIs in NSCLC cells.302 HH signaling pathway drives lung adenocarcinoma (LAC) cells growth under stress conditions such as serum-starvation.303 Moreover, microRNA (miRNA) disorders might be related to cell cisplatin resistance in LAC through targeting Gli2.304 Besides, researchers found that Shh+ NSCLC cells produced full-length Shh protein on the membranes of these cells. Shh+ cells exhibited chemo-resistance and showed proliferation and migration stimulatory effect on Shh- cells in a paracrine manner.305Researchers found that in patients with lung adenocarcinomas tumors and lung squamous cell carcinomas had higher Gli expression, accompanied by significantly lower expression of an EMT marker E-Cadherin. Inhibition of HH signaling pathway in vivo decreased tumor growth and induced E-Cadherin expression.+ hematopoietic cells from patients with chronic myelomonocytic leukemia (CML).307 HH signaling pathway is increased in BCR-ABL+ progenitor cells in CML through enhanced expression of Smo. Smo is responsible for the downregulation of microRNA-326 in patient with CML.309 Inhibiting Gli2 abolishes dormancy in human leukemia stem cells.310It has been found that HH components are aberrantly activated in a variety of hematological malignancies including multiple biological behavior. HH signaling pathway was upregulated in CD34311 In a murine model of myelodysplastic syndrome (MDS), HH signaling pathway activation led to leukemic transformation to AML by acquiring self-renewal potential.312 Stromal cells show potential of supporting activity for proliferation of leukemic cells and the expression of human Hhip in AML/MDS-derived stromal cells was markedly lower than stromal cells from healthy individuals.313 HH/Gli-1 also plays a key role in resistance to radiation, and that inhibition by LDE225 precipitates a radiation-resistant cell line overcoming radioresistance.314 HH signaling pathway has important impact on B-cell in chronic lymphoblastic leukemia, acute lymphoblastic leukemia (ALL) and multiple myeloma.317 HH signaling pathway is associated with progression of these diseases.318 Targeting HH signaling pathway holds promising therapeutic effects.Gli expression can be recognized as a prognostic indicator for acute myeloid leukemia (AML) and elevated Dhh plasma level can be detected in AML patient.319 A majority of Thyroid tumor specimens are positive for HH signaling pathway component  while inhibition of HH signaling pathway reduced proliferation of thyroid tumor cell lines.320 Shh enhances cell motility and invasiveness of anaplastic thyroid carcinomas.321 Moreover, Shh signaling pathway is important to PTCs occurrence and progression.323 It also regulates CSC self-renewal and radiosensitivity in anaplastic thyroid carcinomas cell lines by Snail expression.324 Ma et al. proposed that the loss of primary cilia has been linked to the malignant transformation in thyrocyte.325A total of 80% of thyroid cancers occur as papillary thyroid carcinomas (PTCs).326 Multiple BCC is a major symptom of Gorlin syndrome. 90% of sporadic BCC cases carry somatic mutations in Ptch, others have gain of-function mutations in Smo, leading to over-activation of the pathway.328 Overexpression of Ptch1 and Gli1 were observed in BCC compared with normal epidermal tissue and enhanced Shh immunoexpression was found in the aggressive BCC.330 Interestingly, Gli1 was specifically upregulated in BCC, while the other skin malignancies such as squamous cell carcinoma showed no Gli1 expression.331As the most common skin malignant tumor, BCC is most likely to occur in the skin of head and face.332 The tumor epithelium of human BCC with Smo inhibitor resistance possesses reduced primary cilia. Loss of primary cilia might be related to HH signaling pathway inactivation and upregulated RAS/MAPK pathway in resistant BCCs.333 A lower cilia count in the primary lesion might be correlated with BCC recurrence according to a preliminary study.334 Whitson et al. reported non-canonical hedgehog pathway activation also drives drug resistance of Smo inhibitor in BCC.335Intrafollicular epidermal stem cells are reported to induce BCC formation through HH signaling pathway due to their enhanced self-renewing ability.185 Among all BC subtypes, 10% are triple negative breast cancer (TNBC). Poor prognosis and the lack of efficient targeted therapy make TNBC treatment most challenging.There were 2.3 million new cases of female breast cancer (BC) in 2020, surpassing lung cancer as the most common cancer worldwide.336 HH signaling pathway mediates ductal morphogenesis of the mammary gland during puberty and remains downregulated in normal adult mammary tissue.337Multiple studies have shown that HH signaling pathway plays an essential role in normal mammary gland development.338 Somatic mutations of HH signaling pathway components have been shown to be relatively rare in BC, where ligand-dependent HH signaling pathway activation played a more fundamental role in the pathogenesis of TNBC.339 Moreover, expression of Shh, Dhh, Ptch1, Gli1 has been observed to be upregulated in BC tumor tissue.340 Smo and Gli1 in TNBC tissue is also significantly increased compared to those of non-triple-negative BC.341 Furthermore, TNBC patients with higher Shh expression have worse overall survival.342HH signaling pathway in relation to the aggressive biological behavior of TNBC, including cancer migration, invasion and angiogenesis, has also been discussed.343 Another possible mechanism of TNBC chemoresistance is HH signaling pathway upregulated ATP binding cassette (ABC) transporters and other target genes.338 TNBC cells may also harbor dysfunctional stemness pathways compared to non-triple-negative BC cells.338 TNBC associated fibroblasts can activate HH signaling pathway in a paracrine manner and enhance tumor cell growth.344 In addition, HH-activated CAFs have been shown to form a niche for chemo-resistant TNBC stem cells and target HH-activated CAFs via Smo inhibitors, which benefits patients suffering from TNBC.345 Exposure to chemotherapeutics agents of TNBC cell lines results in the release of Shh and HH signaling pathway activation, leading to an increase in the stemness marker of TNBC stem cells, which may explain the chemoresistance and recurrence of TNBC.346 The above evidence may illustrate one possible way that could relate to the aggressiveness and difficulty in treating TNBC by HH signaling pathway.HH signaling pathway promotes TNBC progression in an autocrine manner, while promoting tumor vascularization via regulation of vascular endothelial growth factor receptor 2 (VEGFR2) expression in a paracrine manner.347 Two major subtypes of this condition are embryonal subtype of RMS (ERMS) and alveolar subtype of RMS (ARMS).348Rhabdomyosarcoma (RMS) is known to be the most common type of soft tissue sarcoma in children whose cells tend to undergo myogenic differentiation.349 The consistent activation of the HH pathway has been associated with RMS, though there have been relatively few RMS cases in patients with Gorlin syndrome.211 According to the Children’s Oncology Group, 18% of patients with fusion-negative RMS harbor a mutation in HH signaling pathway.350The relationship between RMS and HH signaling pathway was first identified in mice heterozygous for Ptch1 as these mutants exhibited a high incidence of ERMS.351 However, activating mutations of HH signaling pathway reported to date only account for a small subset of patients.347 A particular study proposed a different model of RMS in HH signaling pathway activation, where a wide distribution of Dhh and Ihh ligands were detected by immunohistology in RMS samples, in which Shh was only found to be expressed in a small set of RMS cell lines. These findings demonstrated the potential mechanism of the ligand-dependent activation of HH signaling pathway in RMS.352 Hatley et al. utilized aP2-Cre; SmoM2/+ mutant mice and discovered that the activated HH signaling pathway in the adipose lineage led to ERMS in mice.353 Fu et al. reported that primary cilia play a central role in muscle differentiation. Either ciliogenesis or HH signaling pathway are dysregulated in RMS.354Controversy is still prevalent pertaining to the exact mechanism of the constitutive activation of HH signaling pathway in RMS. Overexpression of HH signaling pathway components, such as Gli1 and Ptch1, has been detected in RMS patient samples and human RMS cell lines.356 Meanwhile, upregulation of Gli1 contributes to drug resistance.357Targeting HH signaling pathway in RMS has demonstrated therapeutic potential in in vitro studies, and its inhibition contributes to the suppression of cell invasion and self-renewal in RMS cell lines.However, the pathogenesis of RMS as well as the oncogenic role of HH signaling pathway remains unclear, and further studies should be urgently conducted in order to develop effective treatment options for RMS185 Although the incidence of GC decreases annually, gastric cancer continues to be fatal, with a sharp shortened survival.358Gastric cancer (GC) deaths ranks fourth among all cancers and has led to more than 760,000 deaths in 2020.359 Shh is highly expressed in gastric parietal cells for gastric acid and gastrin production, illustrating that HH signaling pathway is indispensable for normal digestive function.360In stomach, HH signaling pathway is essential for differentiation and maturation of gastric epithelial cells under physiological conditions.361Helicobacter pylori (H pylori) infection is one of the main causes of chronic gastritis and is a significant risk factor for gastric cancer. Multiple studies have reported that chronic H pylori infection can interfere in the balance of HH pathway activity within gastric tissue.360H pylori may also contribute to gastric atrophy and intestinal metaplasia for increased potential of tumorigenesis.362 Mice with parietal cell-specific deletion of Shh were not observed to develop gastritis when infected by H pylori, and Shh in gastric mucosa was considered to act as a chemoattractant for macrophages.363HH signaling pathway links progression from chronic inflammation to cancerous lesions.364 Specifically, Shh expressing in H pylori positive patients with early GC was noted to be significantly higher than H pylori negative controls.365 The increased expression of Shh and Gli1 was also shown to be significantly correlated with tumor staging and tumor aggressiveness, suggesting a worse overall survival for patients suffering from GC.366Shh has been found to be overexpressed in resected GC samples compared to that of adjacent normal tissue.Shh promotes gastric cancer cell proliferation and the survival of lines, which suggests an autocrine progression of gastric cancer. It also regulates gastric cancer migration and invasion of gastric cancer cell lines through EMT.+ gastric cancer stem cells, which was responsible for drug resistance in advanced GC. Moreover, Gli interacts with the promoter of ABCG2 and regulates its expression in gastric cancer stem cells.367 Accordingly, targeting HH signaling pathway may improve GC chemosensitivity.369 Koh et al. investigated PDL-1 expression mediated by Gli in gastric cancer organoids, which may relate to immune evasion in GC.370Shh-Gli1 signaling was activated in CD44371 In light of the rapid understanding of tumor biology, additional features of HH signaling-associated cancers have been determined. Cancer stem cells and the hedgehog pathway are closely related to cancer relapse and cancer resistance to anticancer therapy. In addition, the mechanism of HH signaling pathway activation in cancer is multifactorial, demonstrating that canonical pathway activation and noncanonical pathway activation may exist in a single form of cancer.372 Similarly, ligand-dependent and ligand-independent activation may promote cancer progression cooperatively. Therefore, further studies should focus on the precise mechanism of HH signaling pathway in cancer for better therapeutic targets.HH signaling pathway participates in multiple functions in a variety of tumors. According to immunohistology, early exploration of HH signaling pathway was concentrated on the expression and distribution of the HH pathway components in tumor tissue. One potential reason for controversies in early studies that examined the expression patterns of HH signaling pathway proteins in cancer could be differential tumor samples analyzed.Recent breakthroughs have revealed the carcinogenic function of HH signaling pathway, which makes it an interesting target for cancer treatment. Drugs related to HH signaling pathway target Hhat, Shh, Ptch, Smo, Sufu and Gli1 Fig. . These d373 ATO not only inhibits HH signaling pathway, but also targets other signaling that involved in the development and progression of APL. The first Food and Drug Administration (FDA) approved drug specifically designed to target HH signaling pathway is vismodegib (GDC-449), which was approved for the treatment of BCC in 2012.374 Since then, sonidegib and glasdegib have been approved for clinical treatment of cancer.376 Although vismodegib and sonidegib have achieved satisfactory outcome in the treatment of locally advanced or metastatic BCC, frequent Smo mutations lead to increased drug resistance.377 Therefore, recent studies have revealed that new HH signaling pathway inhibitors and reasonable multi target drugs are applied together to solve this problem, including taladegib, LEQ-506 and TAK-441, which are not sensitive to drug-resistant D473H mutations.380 In addition, some studies on Hhat, Shh and Gli1 inhibitors are also expected to inhibit Smo resistant cells.In 2000, the first drug targeting HH signaling pathway, arsenic trioxide (ATO), was approved for the treatment of acute promyelocytic leukemia (APL).381 Inhibitors targeting Hhat may therefore block HH protein-mediated pathway activation. Current inhibitor targeting Hhat was RU-SKI 43. Petrova et al. carried out a high throughput screen using a peptide-based assay and identified Hhat specific inhibition by RU-SKI 43  both intracellularly and in vitro.382 They further investigated the potential effect of RU-SKI 43 in cancer. The results suggested that RU-SKI 43 was able to reduce Gli1 activation, Akt and mTOR pathway activity and tumor growth in vitro in pancreatic cancer cell proliferation and in vivo in xenograft models by inhibiting Hhat.383 It should be noted that the inhibition effect of RU-SKI 43 was not mediated through Shh protein.383 Another study showed similar observations that RU-SKI 43 treatment reduced the proliferation of estrogen receptor positive breast cancer cells, showing a dose-dependent effect.384 Overexpression of Hhat protein was able to rescue this inhibition, but the addition of exogenous recombinant Shh protein did not.384 The lack of Hhat inhibitor studies may be due to the need to use radiolabeled fatty acid substrates to measure activity, which has limitations in terms of throughput, cost and safety.385 Recently, Andrei et al. proposed an acylation-coupled lipophilic polarization induction assay. They applied this method to discover the potent Hhat inhibitor IMP-1575.386 Future studies will be needed to reveal the role of IMP-1575 in anticancer therapy.Hhat catalyzes the completion of palmitoylation of HH proteins to maintain their stability and normal activity.Shh proteins bind to and inhibit Ptch1 receptor, which could unblock Ptch1 for Smo proteins and cause typical HH signaling pathway. Inhibitors targeting Shh proteins may therefore block Shh protein-mediated signaling pathway activation. Inhibitors targeting Shh include 5E1, Hedgehog-interacting protein (Hhip) and Robotnikinin.387 Treatment of colorectal cancer cell lines with 5E1 reduced the expression of HH signaling pathway target genes  as well as cell cycle protein D1 and mediated the reacquisition of epithelial-like features in cells.388 5E1 was also shown to inhibit the self-renewal capacity and chemoresistance of gastric cancer stem cells, demonstrating a potential role for 5E1 in enhancing chemotherapeutic sensitivity.389 In the non-Smo protein-mediated HH signaling pathway, 5E1 may have a more satisfactory effect. Both cyclopamine and 5E1 could inhibit chronic lymphocytic leukemia (CLL) cells in B-CLL patients.390 Stromal cells could induce activation of the paracrine pathway and activated ERK directly via Ptch1 receptor in CLL cells without Smo.390 In this case, cyclopamine treatment was suboptimal, while 5E1 could completely eliminate ERK phosphorylation.3905E1 is a monoclonal antibody targeting Shh and blocks HH signaling pathway by competing with Ptch1 for the Shh binding site.391 Abnormal expression of Hhip has been associated with cancer development. For example, hypermethylation of the Hhip gene promoter has been found in several tumor cell lines, including HCC, pancreatic cancer, MB, and various gastrointestinal tumors.395 Down-regulation of Hhip expression using siRNA resulted in a significant increase in colon cancer cell growth and invasion in vitro.395 Therefore, adjustment of Hhip level allows regulation of cancer development. Yu et al. used a lentiviral vector to deliver Hhip. The result showed that gastric cancer cells in the Hhip group had reduced proliferation, migration and invasion compared to the control.396 Interestingly, Hhip overexpression significantly reduced its de novo promoter methylation level in gastric cancer cells.396Hhip is a negative regulator of HH signaling pathway that prevents Ptch1 from binding Shh by binding the cholesterol fraction covalently linked to Shh through the N-terminal.397 Robotnikinin reduced Gli2 mRNA levels in human keratin forming cells and in primary human synthetic skin tissue.397 There is currently lack of other studies revealing the potential application of robotnikinin in the treatment of cancer.Robotnikinin is a small molecule that targets Shh-N protein. It has the following features: inhibits HH signaling pathway in a concentration dependent manner; does not show inhibitory activity in the absence of Ptch1 receptors; does not compete with cyclopamine.398 Ptch protein silencing or gene deletion is related to the development of many cancers, such as odontogenic keratocyst, BCC and plexiform fibromyxoma.400 Therefore, activators targeting Ptch can prevent cancer development by inhibiting Smo protein. At present, some natural compounds have been found to increase Ptch1 expression, for example, the combination of epigallocatechin gallery (EGCG) and theaflavin (TF) can reduce the expression of Gli1 and Smo while increase the expression of Ptch1 in mouse liver cancer cells.401Ptch is an inhibitor of HH signaling pathway. Without Shh ligand, Ptch plays a regulatory role by inhibiting the Smo protein.Within HH signaling pathway, Smo protein is often utilized as the drug target because of (1) the ability to transduce downstream signaling pathways in cancers with a loss of function of Ptch1, (2) cancer-causing capacity in the presence of aberrant expression, and (3) the hydrophobic structural transmembrane helical domain capable of binding a variety of small-molecule drugs. The inhibitors targeting Smo mainly include cyclopamine, and cyclopamine derivatives such as vismodegib, sonidegib, etc.Veratrum californicum, which has been shown to bind and inhibit the activity of Smo proteins.403 Cyclopamine may inhibit the development of several cancers by affecting HH signaling pathway.  Cyclopamine is able to block HH signaling pathway in vitro, which in turn affects the aggressiveness and motility of human HCC cells.405 On the other hand, in an in vivo study, Jeng et al. observed a statistically significant reduction in tumor size in mice with HCC (0.152 ± 0.219 cm2 vs. 0.003 ± 0.009 cm2) following a 2-week cyclopamine injection at a dose of 30 mg/kg/day.406 Cyclopamine has also been shown to inhibit the self-renewal and invasive capacity of cancer stem cells in pancreatic cancer and glioblastoma.408 Unfortunately, the poor oral bioavailability and specificity of cyclopamine limit its applications.409 Under this circumstance, semi-synthetic cyclopamine derivatives such as vismodegib, sonidegib and saridegib have been developed, which show better oral bioavailability and perform well in several clinical trials. Of note, cyclopamine may still function as an anti-cancer agent for sensitization to radiotherapy. For instance, Tsai et al. showed that cyclopamine in combination with radiation therapy resulted in a 67% reduction in the average size of in situ tumors compared to radiation therapy alone.410Cyclopamine is a steroidal alkaloid metabolite produced by 411 Vismodegib achieved satisfactory results in clinical trials in patients with locally advanced and metastatic BCC. After 21 months of daily oral administration of 150 mg vismodegib, the objective remission rate was 47.6% for locally advanced BCC and 33.3% for metastatic BCC, with a median remission and progression-free survival of 9.5 months months.413 Several phase I and phase II studies have investigated the use of vismodegib in detail, for instance, vismodegib in the treatment of TNBC, multiple BCC, recurrent/refractory MB, and keratinized cystic odontogenic tumors with significant therapeutic benefit.416 In 2016, Jacobsen et al. evaluated the effect of vismodegib for BCC by meta analysis.417 Data from eight studies with a total of 744 patients showed that: objective response to vismodegib for locally advanced BCC had a weighted average of 64.7% and complete response averaged 31.1%. The objective response for metastatic BCC was 33.6% and complete response averaged 3.9%.417 This meta-analysis showed that vismodegib was identified to have a significant and consistent effect on the median duration of therapy (35.8 weeks) of locally advanced BCC and metastatic BCC. Vismodegib, on the other hand, was not effective in the treatment of metastatic pancreatic cancer, lymphoma, chronic leukemia, and advanced osteosarcoma.420 Therefore, more comprehensive clinical trials are needed to validate vismodegib in different populations. Several clinical trials of vismodegib are currently underway as monotherapy as well as in combination therapy for leukemia, meningioma and chondrosarcoma  of 50.1% for sonidegib compared with 68.5% for vismodegib.427 This indicates that the majority of patients receiving sonidegib and vismodegib achieved promising treatment outcomes. In recent years, some clinical studies have focused on applying sonidegib to other cancers, such as MB, ovarian cancer, breast cancer, etc.430 Sonidegib showed ideal therapeutic effect and safety in the treatment of these cancers. Sonidegib related adverse events included muscle spasms, taste disorders, nausea, alopecia, and elevated creatine kinase levels.431 These reactions are often mild, but long-term adverse reactions may also lead to the decline of patients’ quality of life and drug withdrawal.431 Compared with vismodegib, patients receiving sonidegib treatment had lower overall adverse events and occurred more slowly.432 At present, several clinical trials of sonidegib as a single therapy and a combination therapy are under way, mainly for the treatment of MB (Table Sonidegib (LDE225) is a Smo antagonist found by high-throughput screening in vitro.433 In vivo and in vitro studies show that saridegib can effectively inhibit the proliferation and invasion of chondrosarcoma, serous ovarian cancer, osteosarcoma, acute lymphocytic leukemia and other cancer.437 It is worth noting that saridegib also shows a prominent inhibitory effect on the drug-resistant cells obtained by D473H point mutation after the treatment of MB with vismodegib.438 A phase I study used saridegib and FOLFIRINO  to treat 15 patients with advanced pancreatic cancer. Four out of five patients treated with saridegib monotherapy observed a continuous decrease in CA19-9 (26.9–97.7%).439 In addition, saridegib and cetuximab also showed anti-tumor activity in the phase I study on the treatment of recurrent/metastatic head and neck squamous cell carcinoma.440 The common adverse reactions of saridegib treatment are fatigue, nausea, muscle spasm, liver dysfunction and alopecia.441 These adverse reactions are similar to other HH signaling pathway inhibitors and may be caused by inhibition of the same pathway.Saridegib  is a semi synthetic derivative of cycloparamide in Smo inhibitor, which has significantly improved drug characteristics, efficacy and good pharmacokinetic characteristics.442 A randomized phase II clinical trial showed that compared with cytarabine alone, glasdegib combined with cytarabine can effectively treat AML patients who are not suitable for intensive chemotherapy or have high-risk MDS , with better safety and tolerance.443 Based on this study, FDA approved the combined treatment of glasdegib and low-dose cytarabine for newly diagnosed AML patients who are not suitable for intensive induction chemotherapy in 2018.376 In addition, the phase I study of glasdegib in patients with advanced solid tumors shows that it can keep the patient’s condition stable for a long time.444 At present, glasdegib is conducting further clinical trials, including evaluating whether it can effectively treat AML, MDS or CML patients who have not been treated before with or without cytosine arabinoside (NCT04842604) and whether the combination of glasdegib and temozolomide can effectively treat glioblastoma (NCT03466450). Further clinical research will help to reveal the safety and effectiveness of glasdegib in cancer treatment.Glasdegib is a selective small molecule inhibitor that binds to Smo protein.58 The effect of taladegib is not affected by D473 mutation, and it can effectively treat tumor cells resistant to vismodegib.378 Phase I clinical trial proved that taladegib has acceptable safety in the treatment of locally advanced and metastatic BCC patients. The common adverse events are taste disorder, fatigue, nausea and muscle spasm.445 It is worth noting that another phase I study showed that Notch inhibitor crenigacestat combined with taladegib was poorly tolerated in the treatment of patients with advanced or metastatic solid tumors, showing disappointing clinical efficacy.446 The reason why the combination therapy is not ideal may be due to the heterogeneity of the patient population or the history of systemic therapy.446 Further research on taladegib single drug therapy or combination therapy will fully reveal its safety and therapeutic effect.Taladegib binds to the extracellular end of the Smo transmembrane spiral bundle to inhibit the transmission of HH signal transduction.447 XL-139 can inhibit amyloblastoma cells with SMO-L412F mutation resistant to vismodegib.448 XL-139 combined with gemcitabine can significantly inhibit the growth of CCC in the subcutaneous xenotransplantation model of mice.276 Two clinical studies were designed to explore the effect of XL-139 combined with dasatinib in the treatment of leukemia, but the trial failed to proceed as expected . Therefore, further research is needed to reveal the safety and effectiveness of XL-139.XL-139 (BMS-833923) is a Smo inhibitor, which has been proved to inhibit tumor in many in vitro and in vivo studies. For example, XL-139 can reduce the activity of HH signaling pathway, reduce cell proliferation, while induce apoptosis of esophageal cancer cells in vitro.379 In addition, in vivo study has proved that LEQ-506 at 1% concentration can effectively inhibit the expression of Gli1 (80–90%) in the skin cells of depilated mice.449LEQ-506 is a small molecule inhibitor of Smo, and there are few relevant studies at present. For D473H mutant cells resistant to vismodegib and sonidegib, LEQ-506 was not affected and showed inhibitory effect.450 Similar to LEQ-506, TAK-441 can be used to treat D473H mutants resistant to vismodegib.380 In vivo studies have proved that TAK-441 can inhibit Gli1 mRNA expression and tumor progression in castration resistant PCa and xenotransplantation of pancreatic tumor in mice.452 A phase I clinical trial used TAK-441 to treat 34 patients with solid tumors, including colorectal cancer (26%), BCC (21%) and pancreatic cancer (9%). The results showed that TAK-441 was well tolerated and had preliminary anti-tumor activity.453 The common adverse reactions of TAK-441 during treatment are taste disorder, fatigue, nausea and muscle spasm, mostly mild to moderate.453TAK-441 is an oral small molecule Smo inhibitor. Preclinical studies showed that its 50% inhibitory concentration (IC50) on Gli1 transcriptional activity was 4.4 nmol/L.454 In 2010, it was found that itraconazole could inhibit HH signaling pathway by preventing the accumulation of Smo in primary cilia.455 The unique mechanism of action makes itraconazole a feasible choice for the treatment of other HH signaling pathway inhibitors resistance (such as vismodegib). Many in vivo and in vitro studies have proved that itraconazole can effectively suppress the proliferation of cancer cells, including MB, oral squamous cell carcinoma, gastric cancer, malignant pleural mesothelioma, etc.458 Phase II clinical trial showed that after treatment with itraconazole in 19 patients with basal cells, cell proliferation decreased by 45% (P = 0.04), HH signaling pathway activity decreased by 65% (P = 0.03), and tumor area decreased by 24%.459Itraconazole is a drug to treat systemic fungal infection, especially in patients with low immune function and cancer.461 When inhibiting the proliferation of rat cerebellar granule cells by more than 50%, the required dose of MRT-92 was 0.3 μM and 3 μM for vismodegib.461 Further studies showed that MRT-92 could inhibit the growth of melanoma cells in vitro and xenograft melanoma in mice by inducing DNA damage and G2/M cell cycle arrest.462 Another study identified a new BRD4-SOX2 transcription complex, which is related to the non-standard activation of Gli1 in melanoma.463 The researchers combined the powerful BRD4 degrading agent MZ1 with MRT-92, showing a synergistic anti-proliferation effect on melanoma cells.463 These studies have demonstrated the potential of MRT-92 single drug therapy and multi-drug therapy for melanoma. More studies will help further reveal the safety and efficacy of MRT-92.MRT-92 is a small molecule inhibitor based on acyl guanidine or acyl thiourea scaffold, which can bind to the whole 7-transmembrane domain of Smo, and is insensitive to human D473H.464 PF-5274857 has potential in the treatment of brain tumors driven by the activated HH signaling pathway or brain metastasis of primary tumors, but there is still a lack of further research.PF-5274857 is an oral Smo antagonist. In vivo studies have shown that it can effectively penetrate the blood-brain barrier and inhibit the activity of Smo protein in the brain of mice with primary MB, thereby improving the survival rate of animals.465 Although Smo inhibitors have made good progress in the treatment of cancer, the drug-resistant mutations that they cause cannot be ignored.466 Studies have shown that inhibitors targeting Gli proteins are effective against primary and secondary drug resistance induced by Smo inhibitors.467 Currently, inhibitors targeting Gli include GANT compounds, ATO-like compounds, and Hedgehog pathway inhibitors (HPIs).The nuclear transcription factor Gli is a downstream protein of HH signaling pathway, and inhibitors that target the Gli protein may block its binding to downstream genes and exert an inhibitory effect on HH signaling pathway.468 GANT58 and GANT61 have no effect on other cancer signaling pathways , and therefore they are highly selective for Gli.468 GANTs can inhibit the proliferation of a variety of cancer cells. For example, GANT58 inhibited spheroid formation and invasion of cervical cancer stem cells undergoing EMT.469 Moreover, it showed more potent cytotoxic effects in the face of cyclopamine-resistant T-ALL cells.470The first inhibitors GANT58 and GANT61, which can inhibit Gli protein production, were identified by representative screening in HEK293 cells transiently expressing Gli1 and Gli-dependent luciferase reporter.475 Similar findings were found in in vivo studies, for example, Chang et al. found that the mean volume of cervical cancer in mice after 28 days of GANT61 treatment was significantly reduced compared to the control group (1467.39 ± 403.4 mm3 Vs 460.73 ± 91.01 mm3).476 In addition, GANT61 also inhibited the growth of PCa, MB and rhabdomyosarcoma in vivo.478 Currently, an increasing number of studies are using GANT61 as a positive control for identifying Gli-specific drugs. Notably, GANT61 under physiological conditions is unstable and rapidly hydrolyzes to aldehydes (GANT61-A) and diamine derivatives (GANT61-D).479 GANT61-A lacked biological activity on HH signaling while GANT61-D could inhibit Gli transcription.479 This nature limited the applicability of GANT61 and no clinical trials have been conducted so far.Compared to GANT58, GANT61 has a significant inhibitory effect on more types of cancer cells. For example, in vitro studies have shown that GANT61 can inhibit the proliferation and invasiveness of breast cancer, head and neck squamous cell carcinoma, HCC, ovarian plasmacytoma, and PCa.480 ATO exerts its therapeutic effects by inducing promyelocytic leukemia-retinoic acid receptor multimerization, sumoylation and proteasomal degradation.481 In 2010, ATO was validated as a Gli inhibitor, which inhibited Gli transcription and reduced target gene expression by acting directly on the zinc finger structural domain, thereby preventing Gli accumulation in primary cilia and affecting HH signaling pathway.483 ATO, on the other hand, does not affect the interaction between Gli and DNA.484 Yang et al. showed that after 28 months of single ATO treatment, 39 APL patients (86.67%) achieved complete hematological remission, and these patients showed significant downregulation of Gli2 and Smo gene expression.485 In addition, several in vitro studies have shown that ATO can inhibit pancreatic cancer, osteosarcoma, rhabdomyosarcoma, malignant pleural mesothelioma and MB cells by acting on Gli.488 ATO has been used as a first-line agent in the treatment of APL, but its clinical use in solid tumors is limited by a number of factors, including severe side effects, low drug solubility, and rapid renal clearance.489 Pharmacology, drug combinations, and nano-drug delivery systems have all been effective in increasing the therapeutic potential of ATO.490ATO is an arsenic compound that has been approved by the FDA as a first-line drug for the treatment of APL, with fewer metastases and longer survival after treatment.491 Darinaparsin has better antitumor activity and less systemic toxicity than ATO, and intracellular arsenic levels increase faster, higher, and more consistently than ATO.494 Furthermore, compared to ATO, darinaparsin showed significantly higher in vitro cytotoxicity and radiosensitizing activity against solid tumor cells under normoxia and hypoxia without affecting normal bone marrow.495 Studies have shown that darinaparsin inhibits the proliferation of cancer cells and the growth of prostate tumors in mice by inhibiting Gli2 transcription.496 Phase I clinical trials of darinaparsin for refractory solid tumors and phase II clinical trials of Hodgkin’s lymphoma have shown satisfied performance.498 However, in vivo safety of darinaparsin still requires further study.The toxicity of ATO limits its clinical use in solid tumors, so researchers have developed new arsenic analogs to address this issue. Darinaparsin  is an organic small molecule arsenic compound, and like other arsenic compounds, the arsenic in darinaparsin can bind glutathione and act on the zinc finger structure of Gli protein.499 Each HPI has a unique mechanism of action: (1) HPI-1 inhibits the activation of HH signaling pathway induced by Sufu deletion or Gli overexpression, suppresses Gli protein modification, and affects its function as a transcription factor/cofactor; (2) HPI-2 is less effective against exogenous Gli1 but may interfere with the conversion of full-length Gli2 into a transcriptional activator; (3) HPI-3 inhibits the formation of activated Gli2; (4) HPI-4  acts by disrupting ciliogenesis without affecting Gli2 formation.499 In vitro studies have shown that HPI-1 can effectively inhibit the proliferation of small cell lung cancer, breast cancer and other tumor cells.501 In addition, Wei et al. showed that HPI-4 treatment of chondrosarcoma cells significantly decreased the expression of ciliary microtubule transport protein Ift88, Gli protein, and Ptch1 protein, and impaired cell proliferation and invasion ability.502 However, the low bioavailability of HPI in vivo due to its high lipophilicity and poor water solubility has limited further studies.503HPI is a class of small molecules that directly antagonize Gli, independent of PI3K-AKT, PKA, MAPK and other related pathways.504 It has been found that many natural compounds can target HH signaling pathway and inhibit the proliferation of cancer cells.504 Among them, vitamin D3, also known as cholecalciferol, is the most studied and it is formed by 7-dehydrocholesterol after dehydrogenation of cholesterol by ultraviolet radiation. The A-ring of vitamin D3 can directly bind to Smo and inhibit HH signal transduction.505 Studies have shown that vitamin D3 can reduce the expression of Gli1 and Ptch and cell proliferation in mouse BCC cell line, and this inhibition is carried out in an independent manner of vitamin D receptor.506 At present, several clinical phase I and phase III trials of vitamin D3 are under way to study the role of vitamin D3 monotherapy or combination therapy in non-Hodgkin’s lymphoma and BCC . Since the function of these natural drugs targeting HH signaling pathway has not been fully revealed, we did not include the discussion of these drugs in the current review. Detailed information on natural medicines can be obtained from Table In addition to small molecule inhibitors, there are several inhibitors that can target HH signaling pathway, such as natural compounds and LncRNA. Natural compounds are easy to apply, highly available and acceptable therapeutic methods, usually targeting multiple signaling pathways.507 LncRNA can regulate HH signaling pathway through activation or inhibition, which is related to tumor initiation or progression, chemotherapy resistance, recurrence and other processes.508 The diagnosis or treatment methods based on lncRNA are promising. In Table 509 It is hoped that with the progress of research, more ncRNAs can be approved for clinical treatment.Non-coding RNA (ncRNA) refers to RNA that does not encode proteins and operates at the RNA level. Recent studies have shown that ncRNA can regulate HH signaling pathway by changing Gli1/2, Smo and Ptch1, such as lncRNA, miRNA and circular RNA (circRNA).At present, the selective Smo inhibitors vismodegib and sonidegib have been approved by FDA for clinical treatment for cancers. However, the acquired drug resistance of cancer patients to vismodegib proves the clinical limitation of targeting Smo. Therefore, new inhibitors of HH signaling pathway have been developed to overcome this drug resistance, such as taladegib, LEQ-506 and TAK-441. Their effectiveness and safety need to be further validated in clinical trials. Furthermore, some natural compounds can affect HH signaling transduction through Smo, Gli1, Sufu and related factors. For example, vitamin D3 has entered clinical trials to evaluate its effect on non-Hodgkin’s lymphoma. Yet, these natural compounds have not been proved to be direct targets of HH signaling pathway. This can be further determined by using detailed in vivo and in vitro models. In addition, the bioavailability of many natural products is relatively low, and the concentrations used in in vitro studies cannot be achieved under physiological conditions. Therefore, it is necessary to determine the blood level at which natural products act. NcRNAs can mediate HH signaling pathway by changing Gli1/2, Smo and Ptch1 expression. Nanobased drug delivery systems are developed to deliver oligonucleotide inhibitors, such as liposomes, exosomes, nanoparticles and viral vectors. With the progress of these studies, it will be more conducive to targeted treatment of cancers with ncRNA. Finally, HH signaling transduction pathway also cooperates with other carcinogenic pathways, such as PI3K, MAPK, KRAS/BRAF and TGF-β, etc. It is beneficial to use HH signaling pathway inhibitors as combination or adjuvant therapy in cancer. Existing clinical trials have focused on the combination of HH signaling pathway inhibitors with targeted drugs of other pathways. As such, a future goal should be to define the specific function of drugs targeting HH signaling pathway, including their in vivo effectiveness, safety, side effects, etc. A deeper understanding of the mechanisms of HH-targeted therapies will prompt new treatments and continue to offer potential therapeutic targets for cancer.Early literature primarily focused on defining the function of HH signaling pathway in embryonic development. Recent studies have begun to reveal that the orchestration of HH signaling is critical for healthy cell function. Dysregulation of HH signaling pathway is associated with the development and progression of numerous cancers. Therefore, understanding the integrative nature of HH signaling pathway has opened up the potential for new therapeutic targets for cancer. A variety of drugs targeting HH signaling pathway have been developed, and some of which have been approved as for clinical treatment for cancer. Notably, most research of the inhibitors targeting HH signaling pathway are still in the early stages. The results of several clinical trials are unsatisfactory, owing to the fact that the efficacy and safety of the drugs are not well documented in preclinical studies. Meanwhile, certain drugs targeting HH signaling pathway show limited specificity, which is due to the lack of sufficient studies to verify the mechanism of the specific action on HH signaling pathway. As implied above, with new therapeutic targets being identified at a rapid rate, a more comprehensive understanding of the mechanisms of HH-targeted therapy will solve the existing problems in the application of these drugs and pave the way for a new clinical paradigm.In summary, our review comprehensively summarizes the function of HH signaling pathway in tissue homeostasis and cancer development. Importantly, we discussed the current application, advantages and challenges of HH signaling pathway targeted therapies, providing valuable insight and important implications for the exciting translational innovations in the future.


In recent years, with the increasing incidence of endometrial carcinoma in women of child-bearing age, to decision of whether to preserve patients’ fertility during treatment has become increasingly complex, presenting a formidable challenge for both physicians and patients. Non-fertility-sparing treatment can remove lesions more thoroughly than fertility-sparing treatment. However, patients will permanently lose their fertility. In contrast, fertility-sparing treatment can treat tumors without impairing fertility, but the risk of disease progression is high as compared with non-fertility-sparing treatment. Therefore, it is extremely important to accurately identify patients who are suitable for fertility-sparing treatments. The evaluation of prognostic factors, including myometrial invasion, the presence of lymph node metastases, and histopathological type, is vital for determining whether a patient can receive fertility-sparing treatment. As a non-invasive and quantitative approach, radiomics has the potential to assist radiologists and other clinicians in determining more precise judgments with regard to the above factors by extracting imaging features and establishing predictive models. In this review, we summarized currently available fertility-sparing strategies and reviewed the performance of radiomics in predicting risk factors associated with fertility-sparing treatment. This review aims to assist clinicians in identifying patients suitable for fertility-sparing treatment more accurately and comprehensively and informs more appropriate and rigorous treatment decisions for endometrial cancer patients of child-bearing age.Critical relevance statement: Radiomics is a promising tool that may assist clinicians identify risk factors about fertility-sparing more accurately and comprehensively. Patients who choose fertility-sparing treatment must undergo rigorous evaluations, especially with regard to prognostic factors such as myometrial and lymphovascular space invasion.We found that MRI-based radiomics model performed well in the evaluation of prognostic factors, including myometrial and lymphovascular space invasion.We conclude that the performance of the evaluated radiomics model including both clinical and radiomics features was better than that of the evaluated model that was based on radiomics features only.The respective performances of the evaluated radiomics models need to be comprehensively verified prior to clinical application. Endometrial carcinoma (EC) is one of the most commonly occurring gynecological malignant tumors, and its morbidity ranks sixth among malignant tumors worldwide. According to cancer statistics published in 2020 , the expPatients presenting with different individual characteristics and histopathological features have different profiles in terms of viable treatments and treatment outcomes. Thus, the identification of early-stage patients and the assessment of prognostic factors, including myometrial invasion (MI), lymphovascular space invasion (LVSI), lymph node metastasis (LNM), and cervical stromal invasion (CSI), are critical for evaluating the appropriateness of fertility-sparing treatment –9. As thAs an emerging technology, radiomics can extract image features that cannot be identified by the human eye from imaging. Through processing, image features can be transformed into computer data. Based on these data or combining these data with clinical information, such as clinical features and pathology, it will be possible to develop more valuable diagnosis and prognosis models, which will guide clinicians to make better decision .This review summarizes currently available fertility-sparing strategies and the performance of radiomics models based on magnetic resonance imaging (MRI), ultrasound (US), computed tomography (CT), and positron emission tomography combined with CT (PET/CT) in terms of elucidating predictive prognostic factors associated with fertility-sparing treatments for EC Fig. , includiA search strategy was developed and applied to PubMed, Web of Science, and Scopus. The following words were used for searching:  AND (neoplasm OR carcinoma OR cancer OR tumor) AND (radiomics OR texture)) OR  AND (neoplasm OR carcinoma OR cancer OR tumor) AND (fertility-sparing OR fertility preservation) AND (radiomics OR texture OR imaging)). The search date ends in August 2021. A total of 604 results were searched in three databases; after removing repetition, the articles that are associated with the evaluation of risk factors related to fertility-sparing were included.The selection of eligible patients (among those choosing fertility-sparing treatments) is highly rigorous. The indications recommended by the National Comprehensive Cancer Network , the EurThe standard treatment for women with EC who are of child-bearing age is total hysterectomy and bilateral salpingo-oophorectomy (TH/BSO). This is an effective method with a high five-year survival rate of 93% . HoweverThe most commonly prescribed therapeutic schedule is a standard regimen of high-dose oral medroxyprogesterone. The levonorgestrel intrauterine device can replace oral progesterone in women with complicated atypical hyperplasia . In addiIn addition to selecting appropriate treatments, close follow-up is an important factor in ensuring the safety and efficacy of fertility-sparing treatments. The surveillance strategies recommended by the National Comprehensive Cancer Network are shown in Fig. Moreover, the European Society for Medical Oncology has demonstrated that progesterone receptor status can reliably predict therapeutic response in EC. However, this indicator is not recommended as a routine test, because 50% of progesterone receptor-negative patients have been shown to respond to therapy .Currently, imaging  is one of the most commonly utilized methods for the preoperative assessment of EC –19. MostMRI with good resolution of soft tissues can distinguish endometrial lesions and myometrial signals to clearly show the range of lesions through contrast dynamic enhancement scanning. In recent years, MRI has been used to describe the local extent and expansion of tumors in various studies –23.Moreover, US conducted by imaging experts has a nonnegligible role in evaluating the extent of EC in the pelvis and abdominal cavity. As a non-invasive, cheap, and convenient imaging method, US is appropriate for all patients. Epstein et al. revealed that ultrasonic and Doppler characteristics can be used for risk stratification in EC, and this finding was validated by a subsequent prospective study .In contrast, CT is unreliable for predicting EC stage. This is because the contrast between the tumor and the myometrium is difficult to identify, thus rendering the tumor invisible in CT imaging. Thus, CT cannot be used to assess the depth of MI and CSI . Due to Additionally, PET/CT has been proven to be equal in performance as compared with MRI in terms of predicting MI . HoweverImaging has been widely used in clinical practice. However, the identification of tiny lesions is difficult in practice. In addition, it is difficult for radiologists and clinicians to acquire all imaging information associated with lesions through simple visual evaluations, and the differences between different imaging readers cannot be well controlled. Radiomics has the potential to overcome these problems.As a non-invasive approach, radiomics can extract quantitative and repeatable image features for analysis. Radiomics analysis includes the following six steps.The acquisition of high-quality images is the foundation of radiomics, which has a meaningful impact on effective analyses. The uniformity of imaging machines and protocols should be ensured. However, if this is not possible, biases should be corrected via previously described protocols , 26.The segmentation of images is critical for radiomics analysis. The region used in the subsequent analysis is obtained through image segmentation. For tumor analysis, the focus was the pixels and voxels in target area, which can be either two-dimensional or three-dimensional region. The two-dimensional region usually was the axial section where the maximum dimension of tumor is located and the three-dimensional region usually was the tumor itself. The segmentation method includes manual, semi-automatic, and automatic segmentation. Manual segmentation by an imaging expert is the gold standard for image segmentation , 26–30.Some key elements, including imaging methods, imaging protocols, and segmentation protocols, must be well defined through data preparation, which has a meaningful impact on model creation .Radiomics features are defined as quantitative data extracted from imaging. Radiomic features are divided into four classes: first-order statistics, shape, texture features, and features obtained by wavelet transformation of relevant image sections. First-order statistics features can describe the distributions of voxel intensities. The shape features are associated with the shape of the volume. Texture features can reflect heterogeneity within tumors, including many gray-associated features. Wavelet features calculate the intensity and texture features from the wavelet decompositions of the original image , 27.Relevant features should be selected from among many radiomics features, and alternative features should be removed or transformed. This process is termed dimensionality reduction –31.Establishing a model that can precisely predict the classification or prognosis of a disease is the primary goal of radiomics. There are many methods for establishing a radiomics model. Deep learning is the preferred method when the sample size is sufficiently large , 26, 29.The performance of different radiomics models in predicting prognostic factors associated with fertility-sparing treatment is shown in Tables p = 0.48).Deep myometrial invasion (DMI) is considered the most significant single morphological prognostic factor in patients with EC . Ueno etThe definition of LVSI is that tumor cells are lined by endothelial cells outside the immediate invasive border . The modLNM is an important factor affecting EC prognoses . AlthougCSI is a risk factor for poor prognoses in EC. For early CSI, superficial lesions are often indistinguishable from the cervical mucosa on MRI, and the presence of chronic inflammation of the cervix often interferes with establishing diagnoses. This may in turn influence treatment decisions. If the presence of CSI can be accurately identified prior to surgery, this information would provide critical help for subsequent treatment decisions. However, according to a study conducted by Ytre-Hauge et al., there is no evidence that MRI-based texture analysis is conducive to the identification of CSI when compared with traditional MRI .ADC, which delineated the ROI manually in the apparent diffusion coefficient map; ModelR, which segmented the tumor manually and subsequently extracted radiomics features; and ModelM, which combined radiomics features with information on CA125 and body mass index (BMI). The performance of the ModelADC was limited in the prediction of histological grades. In the training cohort, the AUC of ModelM was higher than that of ModelR. However, the difference between ModelM and ModelR was not statistically significant in the test cohort. This may be attributed to the small sample size of the test cohort. In summary, the three evaluated models presented different levels of prediction. However, we concluded that ModelM, which includes both radiomic and clinical features, was the most effective model by far.Histological grades, a vital prognostic factor, are important for choosing effective treatment strategies. Ytre-Hauge et al. reported that magnetic resonance texture analysis has the potential to predict histological grades in EC, as they found some features that were predictive high-risk histological subtypes . MoreoveA large multicenter study conducted by Yan et al.  collecteWomen suspected of having EC must undergo two tests: transvaginal ultrasound and endometrial biopsy . For theYtre-Hauge et al.  establis18F-FDG PET/CT imaging. A prior retrospective study reported that volume density and irregular shape are the most strongly associated features for predicting LNM using PET/CT and that this method has high specificity for detecting LNM in EC [p value and highest AUC. The sensitivity and specificity in training cohort were 0.75 and 0.81, respectively. In test cohort, the sensitivity and specificity were 0.89 and 0.80, respectively.PET/CT is an imaging method employing tracers, and the most commonly used tracer is fluorodeoxyglucose (FDG). FDG is taken up by cells that use glucose efficiently, such as tumor cells, and is subsequently detected by PET imaging. EC has a high associated rate of glucose metabolism and glycolysis, making it suitable for NM in EC . ElisabeNM in EC  extracteThis review summarizes progress in terms of radiomics-based fertility-sparing treatments in EC. In recent years, with the extension of research and multicenter clinical trials, the prediction performance of EC risk factors has become increasingly accurate. However, some issues and concerns still exist.First, the existence of selection bias might have biased the results of this study. For example, when analyzing the performance of radiomics in predicting DMI, all enrolled patients underwent hysterectomy , 33. To We summarized the performance of radiomics in the evaluation of risk factors related to fertility-sparing. Radiomics is a promising tool that may assist clinicians identify risk factors about fertility-sparing more accurately and comprehensively. However, more research should be done before deploying radiomics into radiologists’ daily practice.