According to the current understanding, cancer tends to be defined as a group of malignant diseases with multiple genetic origins. Therefore, gene mutations cannot be ignored in the occurrence of cancer. In normal tissues and organs, the HH pathway is a crucial pathway for development. For instance, the HH pathway controls the migration of granular neuronal precursors to the correct location in the brain (12). Once mutation of HH occurs, the opportunity for tumorigenesis occurs. According to the 2021 World Health Organization guidelines, medulloblastomas can be divided into four molecular subgroups, of which the evaluation is partially based on HH genotyping (13).

Gorlin syndrome (GS), a rare autosomal dominant disease, is caused by mutations in the HH pathway. The typical mutations observed in GS cause pathogenic variations of PTCH1 and SUFU. In the differential diagnosis of GS, it has been suggested that PTCH2 may be a candidate gene for the prediction of susceptibility, and mutation of PTCH2 has been reported in clinical practice (14). However, a recent study asserted that PTCH2 should not be included in the genetic diagnosis of GS (15). In fact, the PTCH2 gene has different functional characteristics to PTCH1. PTCH2 is considered to coordinate PTCH1 to alter SMO localization and plays an auxiliary role in regulating the HH pathway (12). This may explain the absence of a statistically significant detection of PTCH2 mutations in the clinic.

A recent study indicated that 68% of SUFU pathogenic variation carriers had at least one type of tumor, and the incidence of tumors in their relatives reached 44.1% in individuals up to 50 years-old (16).

SMO mutations have also attracted wide attention due to resistance mutations that prevent effective drug binding (discussed later). In addition, carcinogenic mutation of SMO is widespread. In this instance, the mutation stabilizes the active form of SMO and releases it from the conformational restriction of the inactive state, and thus may induce clonal expansion together with tumor initiation and invasion (17). SMO multi-site mutations have been revealed to be a carcinogenic driver for a variety of epithelial-derived tumors and brain tumors (18).

GLI mutations have also been reported in the clinic. In addition, a study has suggested that upregulation of a GLI2-β subtype lacking the N-terminal repressor domain induced chromosome number and structure aberrations, which disrupt genomic stability (19).

The latest version of COSMIC v99 (released November, 28 2023; http://cancer.sanger.ac.uk) was used to summarize the mutation rate of key molecules in the HH pathway in different tumor types. As demonstrated in Tables I, SI and SII, except for central nervous system medulloblastoma (SHH subtype) and basal cell carcinoma, alimentary tract cancer and adenocarcinoma demonstrated a higher incidence of HH pathway mutation. However, the latter two tumors have not markedly benefited from HH pathway inhibitors. It is considered that some pathway inhibitors affect gene expression due to their diverse treatment mechanism and rich therapy targets, as well as crosstalk with other pathways and epigenetic reprogramming.

In addition to single-gene mutations, multi-gene fusion has also received attention in research. Common gene fusions occur between GLI1 and other genes (Table SII). At present, the MALAT1-GLI1 gene fusion has been identified as one of the diagnostic criteria for gastroblastoma (20). Regardless of the concrete types of multi-gene fusion, inhibition of HH can be used as a general treatment method. Furthermore, the upregulation of GLI1 has been defined as an alternative genetic mechanism for GLI1 fusions, with the characteristics of co-amplification of the cyclin-dependent kinase (CDK) 4 and MDM2 genes (21,22).

In addition to altering the DNA sequence, DNA can also be chemically modified by methylation and hydroxy-methylation to regulate gene expression (23,24). By querying the MethMarkerDB database (https://methmarkerdb.hzau.edu.cn/) (25), the overall degree of methylation of HH pathway-related genes was determined and the potential of differentially methylated regions for the diagnosis (Table SIII) and prognosis (Table SIV) of some cancer types was underscored. Among them, the low methylation level and high expression of GLI1 in melanoma, GLI2 in ocular melanomas and GLI3 in stomach cancer suggested an improved patient prognosis. PTCH1 in kidney clear cell carcinoma and GLI3 in colon and prostate cancer were also considered to be robust diagnostic biomarkers. In addition, clinical data supports the suggestion that promoter methylation is a critical regulatory mechanism of SHH (26), PTCH, GLI (27) and SMO (28) expression. Accordingly, folic acid (29) and DNA methyltransferase inhibitors (27) can effectively block the HH pathway for the treatment of cancer.

Notable heterogeneity in methylation and gene expression levels were also observed in the MethMarkerDB data (Tables SIII and SIV). This heterogeneity may be ascribed to the limited sample size. However, abnormal epigenetic characteristics of cancer cells can also be induced by phosphorylation, acetylation and other acylation modifications of histones in cellular chromatin (23,24). The phosphorylation of GLI1 can be disrupted by mutations in AMP-activated protein kinase (AMPK), which increases carcinogenic potency. As acetylated proteins, GLI1 and GLI2 rely on histone deacetylase (HDAC)-mediated deacetylation to promote transcriptional activation. HDAC-related changes also directly lead to changes in GLI. Therefore, HH and HDAC inhibitors may exert a synergistic antitumor effect (30). Additional modifications include: Sufu negating protein 1 ubiquitinates SUFU via ligand of numb-protein X 1 (31), runt-related transcription factor 3 promotes GLI ubiquitination (10) through the E3 ubiquitin ligase family (32) and protein phosphatase 4 regulatory subunit 2 promotes SUFU dephosphorylation (33).

Recent developments in the research of ncRNA have attracted marked attention, particularly microRNA (miRNA), which plays a regulatory role by directly cutting or preventing the translation of mRNA. In addition, circular RNA, rich with miRNA binding sites, can sponge miRNA to relieve the inhibition of mRNA. The binding of long ncRNAs to RNA-binding proteins confers a variety of regulatory functions, including managing genomic instability. The specific regulation of key molecules of the HH pathway by ncRNAs is summarized in Table II (11,34–127). As summarized in Table SV, the HH pathway can also affect the expression of other pathway molecules by regulating ncRNA to regulate tumor phenotypes.

The HH pathway mechanism in tumor-promoting inflammation can be exemplified by Hp infection, which has been proven to be associated with gastric cancer. Once infected, Hp injects cytotoxic associated gene A into gastric cells, resulting in the accumulation of bone marrow cells in the stomach following the secretion of SHH ligands by parietal cells. Then, these cells differentiate and mature under the induction of factors such as IFN-α, express GLI1-dependent schlafen 4 and secrete interleukin (IL)-1β to activate the IL-6/phosphorylated signal transducer and activator of transcription (STAT)-3 pathway. The activation of SHH during Hp infection was closely related to the expression of programmed death-ligand 1 (PD-L1) (128), as well as the emergence of myeloid-derived suppressor cells (MDSCs), intestinal metaplasia and soluble polypeptide expression metaplasia (129).

The relationship between hepatocellular carcinoma and the HH pathway activated by chronic liver injury has also been clarified. Specifically, hepatitis B virus X protein has been revealed to directly interact with GLI1 and promote disease progression when GLI2 is not inhibited by Sestrin 3 (130).

In pan-cancer, activation of the transcriptional programs of SHH in the non-T cell-inflamed tumor microenvironment was discovered in a study by Bao et al (131), after performing an unbiased genome-wide pathway discovery. Furthermore, non-classical HH activation can promote tumor development by generating pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-1β (132) and TGF-β (133), in hypoxic or inflammatory environments. In addition, hypoxia also contributes to the enrichment of immunosuppressive cells (134), as well as the excessive activation of the Notch/HH axis (135). Notably, STAT is one of the main factors of pro-inflammatory signal transduction. The interaction between STAT and HH is an important component of inflammation and tumor promotion mechanisms (136). In basal cell carcinoma, IL-6 has been revealed to cooperate with carcinogenic HH/GLI signaling via the IL-6R/Janus kinase 2/STAT3 pathway (137). In addition, the antitumor effect of IFN-γ/STAT1 can be antagonized by the activation of suppressor of cytokine signaling 1 by GLI1 and GLI2 (138).

By contrast, the anti-inflammatory effect of HH pathway activation is also a protective mechanism, particularly in acute inflammation. Increased expression of the anti-inflammatory cytokine, IL-10, in HH pathway-responsive stromal cells and concomitant increases in CD4 forkhead box (Fox)p3 regulatory T cells (Tregs) reduces the tumor burden of colitis-associated colon cancer (139). Similarly, the pancreatic gland is also protected from acute pancreatitis by the SHH/GLI1/IL-10 axis (140). When considering that the HH pathway is also involved in the interference of cell differentiation and maturation during tissue repair following inflammation (141), whether to administer pathway inhibitors in the early stages of inflammation and the risk of tumor development remain to be considered.

In most tumors, fibroblasts, endothelial cells and macrophages exhibit strong positive connections with GLI (143). In previous studies, TAMs derived from MDSCs were revealed to secrete a variety of anti-inflammatory cytokines and express immune checkpoint ligands that inhibit effector T cells and recruit Tregs (144,145). Research thus far has mainly focused on the association of TAM aggregation with abnormal expression of SHH and activation of HH/GLI (146–148). With the participation of krüppel-like factor 4 SHH-derived TAM M2 polarization (149), and GLI1 can directly affect the M2 activation state by activating the feedforward loop of STAT6-IL-4ra (150). In addition, the STAT3 pathway has also been revealed to be involved in the regulation of PD-L1 expression in TAMs by tumor-derived SHH ligand (151,152). The carcinoma cell-TAM-carcinoma cell loop may produce a hierarchical amplification effect via the SHH/GLI2-TGF-β1 loop, promoting tumor growth (153). By targeting tumor-supportive M2-like TAMs, a synergistic effect of peroxisome proliferator activated receptor γ with SMO has also been proven (146).

In an infectious state, GLI1 directly increases the transcription of cyclooxygenase-2/prostaglandin E2 and regulates miR-324-5p and miR-338-5p. These miRNAs target PD-L1 to increase its expression, finally realizing the SHH/phosphoinositide-3 kinase (PI3K)/mTOR/NF-κB signal transduction of dendritic cells and activating Treg amplification (154). In gastric cancer, this process was discovered to be mediated by the mTOR signaling pathway, involving the SMO-independent HH pathway (145). In addition, blocking HH reprogramed Treg trans-differentiation into inflammatory Th17 cells, which enhanced the recruitment of cytotoxic CD8⁺ T cells into tumors (155).

HH activity detection can be used to predict the efficacy of immune checkpoint inhibitors, with the support of clinical data (156,157). The expression of programmed death protein-1 (PD-1)/PD-L1 induced by various pro-inflammatory factors changes under the crosstalk of HH and other pathways (147,158,159). The high prevalence of Tregs within the tumor microenvironment and induction of PD-1/PD-L1 is likely to constitute a major mechanism of immunosuppression by HH/GLI signaling in cancer (147). These findings increase the therapeutic opportunities for combination treatments with HH and immune checkpoint inhibitors.

In the unlimited proliferation of tumors, telomerase determines the replication potential of cancer cells. Research has revealed that hTERT is a direct target of HH/GLI, and the effect on hTERT activity is related to differentiating benign and malignant tumors (160). Reduced cell proliferation and GLI1 levels have also been observed in cancer cells with long-term use of telomerase inhibitors (161). However, it cannot be concluded that TERT forms a feedback loop with HH, since the current suggestion is that TERT upstream of HH is more likely to activate the HH pathway by regulating miRNA (162) and recruiting pro-oncogenic transcription factors (163). These effects contribute to increasing invasion and are independent of changes in telomerase activity.

Regulation of the tumor cell cycle is mainly dependent on two important pathways: The RB transcriptional corepressor 1 and p53 pathways. Furthermore, mutations in these two pathways lead to the formation of primary cilia, abnormal elevation of Hedgehog ligands (164,165) and direct regulation of downstream cell cycle regulators, such as cyclin (166) and CDKs (167). It mediates HH-induced DNA replication. This was also partially dependent on the PI3K/AKT pathway (167). The HH pathway has also been revealed to block its inhibition of cyclin regulation by affecting stem cell-related factors, such as BMI-1, to inhibit the downstream p14 and p16 proteins (168). Another well-known cell cycle regulator is FoxM1. GLI1 has been demonstrated to bind to FoxM1 and initiate the effect of Xenopus kinesin-like protein 2 on cell division (169). The crosstalk between the Notch pathway as a proliferation-related pathway and the HH pathway also decreases G1/G0 cycle retardation (170).

The HH pathway has been implicated in regulating apoptosis. TRAIL has become a new target for cancer treatment due to reduced toxicity to normal tissues and specificity for tumor cell apoptosis. The positive crosstalk between NF-κB and the HH pathway amplifies the effect of resistance to TRAIL-related apoptosis (171,172). However, blockade of the GLI family increases TRAIL sensitivity (173).

A considerable number of studies have revealed that the Bcl-2 family are target genes of the HH pathway, including BCL-2 (174), myeloid cell leukemia-1 (174,175), BCL-XL (174) and NOXA (176), which play an anti-apoptotic role by blocking the activity of the caspase family, leading to TRAIL resistance. Bcl-2 proteins also form a feedforward signal with SUFU to further induce the expression of target genes in the HH pathway (174). Accordingly, inhibition of the HH pathway provides a new avenue for the treatment of TRAIL-resistant tumors.

Among the apoptotic pathways, the MYC family (oncogenes related to apoptosis) also closely interact with HH. Upregulation of the family member protein, MYCN, in basal cell carcinomas is a key factor that re-activates dormant HH signals and induces tumor progression (177). Similarly, MYCN has been identified as an invasive marker in neuroblastoma, which was associated with HH signaling, for determining prognosis. Although the positive or negative correlation between GLI1 expression and prognosis in neuroblastoma remains controversial, most evidence suggests that high GLI1 expression indicates an improved prognosis in MYCN-amplified neuroblastoma (178). An explanation for this is that protein kinase-like endoplasmic reticulum kinase-EIF2α pathway is an important mediator in Hh-dependent autophagy on MYCN-amplified neuroblastoma (179).

In addition to TRAIL and the MYC family, the anti-apoptotic effects of other apoptosis-related factors also depended on HH. For instance, TNF-α induces the expression of activator protein 1 family members and regulates apoptosis through the HH pathway (180,181). Survivin, another inhibitor of apoptosis family, has been revealed to be a transcriptional target of GLI (182). HH/GLI1 signaling mediates the RNA polymerase III signaling pathway and tRNA synthesis to regulate the cell cycle and death receptor binding (183).

It is worth noting that the effect of HH-induced autophagy on apoptosis does not act alone in various cancer types. On the one hand, HH-induced autophagy leads to cell death and affects a variety of cancer types by regulating targets such as BCL-2 interacting protein 3 and LC3 II (184,185). Correspondingly, certain drugs have been revealed to induce autophagy and cell death by blocking HH signaling (186,187). On the other hand, HH-induced autophagy can also promote tumors by providing energy for tumor development. SHH inhibition in thyroid cancer activates TAK1, phosphorylates JNK/AMPK and induces autophagy (181). Therefore, acknowledging the effect of autophagy on apoptosis caused by inhibition of HH can guide the usage of drug combinations more reasonably.

In addition to systematic apoptosis pathways, another phenomenon, dependence receptors triggering apoptosis signals without ligands, have been gradually recognized. Autocrine SHH interference in colon, pancreatic and lung cell lines triggers cell death via PTCH proapoptotic signaling (188). The cell-adhesion molecule-related/downregulated by oncogenes protein and its ligand, SHH, perform identically (189). However, very little optimization work has been conducted on this finding.

In addition to interfering with apoptosis, HH has also been implicated in resistance to cell death induced by physical and chemical factors (166). Under radiation, HH is upregulated, which may be driven by TGF-β and TNF-α (190) and mediated by mTOR/ribosomal protein S6 kinase β-1 (191). Atypical protein kinase Cι/λ and GLI can also form a positive feedback loop under high levels of radiation to change the radiosensitivity of tumors (192). In addition, the HH pathway activated by chemoradiotherapy also increases the rate of tumor proliferation by upregulating the G1/cyclin/Rb axis (193). In response to irradiation, GLI1 activates RNA polymerase I, which synthesizes ribosomal RNA for accommodating cell proliferation and division (194). Thus, blocking HH signaling was demonstrated to be an effective method for inhibiting accelerated tumor repopulation following therapy.

Hypoxia is a mechanism underlying tumor invasion and metastasis that is partially achieved by activating the HH pathway. Hypoxia is often accompanied by TNF, NOX4 and other products. TNF-α can upregulate the expression of GLI1 via NF-κB transcription to achieve EMT and drug resistance (132). Furthermore, NOX4 expression triggers the reactive oxygen species-mediated non-canonical HH pathway in the initiation of EMT (198). Hypoxia inducible factor (HIF) is also a widely studied regulator. The upregulation of HIF-1α was revealed to be associated with the upregulation of SHH ligand secretion and GLI1 expression, thereby increasing metalloproteinase (MMP) expression and EMT progression (199,200). It should be noted that activation of the HH pathway during hypoxia leads to an increase in stromal fibroblasts, then the deposition of fibrous tissue and ultimately the aggravation of hypoxia (201). Hypoxia can also provoke HIF-2α to induce GLI1 activation through a SMO-independent pathway (202), which can be ablated by PI3K inhibitor or MEK inhibitor (202). This finding also suggests a complex connection between these pathways.

The crosstalk between the HH, PI3K and MEK pathways has been gradually clarified. Previously, there was evidence that fibroblast metastasis required the stimulation of Ras homolog family member (Rho)A by SMO, which can be realized by a heterotrimeric Gi proteins/PI3K/Rac1 series of activations (7). Subsequently, it was revealed that the PI3K/AKT/mTOR pathway plays an important role in SHH signaling to promote metastasis (203). More specifically, AKT/GSK3β signaling mediates the upregulation of GLI1 expression, thereby obtaining epithelial mesenchymal plasticity (204). In addition, astrocyte elevated gene-1 protein is induced by PI3K/AKT signaling and is vital in the metastasis and development of various types of cancer. Therefore, it was not surprising that PI3K pathway is involved in the crosstalk process (205).

In the MEK pathway, GLI1 directly binds to the promoter region of the CXCR4 gene and participates in stimulated signal transduction of CXCL12 to stimulate the phosphorylation of ERK (206). This is consistent with the observation that HH upregulates MMP-9 expression via the ERK pathway (207), while MEK/ERK signaling is involved in the regulation of GLI1 activity in turn (208,209). Furthermore, the MEK/AKT pathway could be regulated by RAS and others. The widespread occurrence of this regulation in cancer cells rectifies the singleness of the HH pathway, which is mainly activated in the microenvironment but not tumor cells (208,209). The close relationship between these three pathways supports the notion that the synergistic role could be amplified by blocking the pathways simultaneously (210).

The HH and Wnt/β-catenin pathways have also been demonstrated to impose a synchronized regulation on tumor metastasis. In fact, a crosstalk between these pathways does exist (89,211). During the development of cancer associated fibroblasts (CAFs), two pathways regulate the target gene of TGF-β/SMAD3 to participate in the EMT process (212). Although uncertainty remains as to the specific mechanism, it is not difficult to observe that tumor invasion and metastasis are the ultimate outcome of multiple pathways.

In the previous section, the upstream and downstream molecular mechanisms of GLI1 were briefly mentioned. In gastric cancer, galectin-1 from CAFs binds to β1 integrin and targets GLI1 to promote both EMT and vasculogenic mimicry (213). Signal peptide CUB EGF-like domain-containing protein 2 (214) and FoxF1 (215) both regulate tumor metastasis via GLI1. In addition, S100A4 is a newly discovered downstream target gene of GLI1 (216). However, a new theory suggests that tumor progression is dynamically regulated between proliferation and metastasis through up and downregulated GLI1 levels, rather than just upregulated GLI1 alone. Moreover, endogenous GLI1 can directly bind to the promoter of the E-cadherin gene, termed CDH1, and resist EMT (217).

Compared with GLI1, truncated GLI1 (tGLI1) demonstrated a stronger association with abnormal HH signals. In addition to regulating the known GLI1 target genes in the crosstalk with STAT3 and other pathways (218), tGLI1 can also regulate the expression of genes that were not regulated by GLI1, including vascular endothelial growth factor (VEGF) and heparinase (219). Moreover, tGLI1 displays a strong correlation with tumor metastasis. The presence of tGLI1 enhances the expression of MMP-2 and MMP-9 and may target twist and snail (220). In addition, tGLI1 has been identified as a brain metastasis-promoting transcription factor in breast cancer (221). tGLI1 increases the degree of cancer cell stemness by upregulating genes such as Nanog and by activating astrocytes to achieve metastasis (219). In summary, it is hypothesized that tGLI1 is more likely to be a marker for the diagnosis and prognosis of cancer metastasis, and thus may become a new therapeutic target (222).

With the accumulation of research, the unknown role of ncRNA in EMT progress and MMP expression has been gradually uncovered (Table II).

The Warburg effect has been widely discussed in terms of tumor metabolic changes. This effect allows cells to replace the mechanism of oxidative phosphorylation with aerobic glycolysis, thereby obtaining more energy to promote tumor development. The HH pathway mediates the Warburg effect of tumor cells and CAFs. Caveolin-1 (Cav-1) is present in the tumor matrix and participates in the regulation of glycolytic activity (232). The deletion of Cav-1 is associated with high expression levels of GLI1 (233). It was also revealed that the activation of SMO promotes glycolysis via GLI upregulation and the AMPK-mediated activation of hexokinase 2 and pyruvate kinase 2 (234).

In addition, HH activity is involved in the regulation of metabolism and bioenergy in TAMs. Inhibition of HH causes metabolically demanding M2 macrophages to shift their metabolism and bioenergetics from fatty acid oxidation to glycolysis (148). HH signaling also acts downstream of metabolic reprogramming to influence tumorigenesis. HH signaling mediates the hyperglycemia inducing glycolytic phenotype and promotes EMT via Yes-associated protein 1 (235).

Ornithine decarboxylase (ODC)1 is aberrantly upregulated in primary (SHH subtype) medulloblastoma, which increases polyamine metabolism to promote tumors (236). In this instance, AMPK promotes the stable formation of the SUFU/CCHC type nucleic acid binding protein (CNBP) complex via phosphorylation of CNBP, and further promotes the expression of ODC (237). In addition, a metabolic switch to oxidative phosphorylation was revealed to be promoted by GLI1 editing (238).

The use of HH pathway inhibitors as anticancer drugs has gained significance. At present, five HH pathway inhibitors have been approved for marketing. A large number of new generation inhibitors and drugs targeting new targets have entered clinical trials. Among them, SMO inhibitors are classic inhibitors as inhibition of SMO is a reliable route to blocking activation of the HH pathway. However, the frequent occurrence of drug-resistant mutations in SMO means that traditional SMO inhibitors are prone to failure, a problem that requires urgent attention. Second-generation SMO inhibitors have been proven to be effective in preclinical experiments by targeting specific SMO site mutations or by improving the binding affinity. Directly targeting the downstream signals is another traditional solution. The HH inhibitor drugs are summarized in Table III.

HH pathway mutations have been revealed to be associated with enhanced tumor immunogenicity, and it may be feasible to further seek immune checkpoint inhibitor treatment for greater benefits (239). SMO resistance can also be prevented by altering the epigenetic changes of histones or transcription factors GLI1 and GLI2, such as by HDAC (240). Moreover, new technologies such as CRISPR/Cas9 for pathway site-specific gene editing are also being developed (241), which may pave the way for HH therapies. Although drugs for downstream targets are under study, progress has been slow (242), which may be partly due to downstream signals, such as GLI1, being activated by other pathways, such as the TGF-β, Ras and PI3K/AKT pathways. Therefore, blocking the activity of non-classical pathways is also being considered as a new alternative strategy (243).

Drugs targeting ncRNA should also be considered as a future direction for HH pathway-related treatment. However, how to narrow the scope of the best target ncRNAs and how to improve recognition of the structure of ncRNA remains to be solved.

As aforementioned, a variety of anti-HH/GLI drugs have been developed. However, the complex crosstalk between pathways, compensatory mechanisms, the generation of primary or secondary drug resistance, as well as toxic side effects, may still lead to the failure of current drugs. Recently, natural drugs with multiple targets and a higher safety profile have attracted attention. The combination of natural drugs and anticancer drugs has been revealed to be more effective than anticancer drugs alone. In Table IV, the new progression of some natural components and their main targets and effects in research, which may provide support for the future transformation of natural drugs based on the HH pathway into anticancer drugs, are summarized.