The Hedgehog (Hh) gene was discovered in 1980 by Nusslein Volhard and Wieschaus for genetic analysis of Drosophila melanogaster. In the early 1990s, three Hh homologous genes were found in vertebrates, namely Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh). Dhh and Ihh play important roles in the development of normal tissues such as pancreas, testicular formation, and bone development. Shh is the most important member of the three and is widely expressed in adult organizations.
The Hh signaling pathway is a highly compliant signaling pathway that plays an important role in many aspects. Especially, it plays a vital role in embryonic development and growth regulation, being morphogens, mitogens and organ development inducing factors, such as cell proliferation, cell adhesion, cell migration, cell differentiation and embryogenesis. Hh signaling pathway can participate in wound healing of multiple mature organ tissues simultaneously and is one of the important factors involved in liver damage repair, but its excessive activation can lead to liver fibrosis and liver cancer. Shh has been shown to be a strong oncogene, and overexpression of Shh induces basal cell carcinoma in mice. Excessive activation of the Hh signaling pathway has been detected in a variety of solid tumors, such as medulloblastoma, leukemia, breast cancer, prostate cancer, pancreatic cancer, basal cell carcinoma, non-small cell lung cancer, and small cell lung cancer. Statistics show that up to 25% of human cancer deaths may be associated with abnormal Hh signaling pathways. Therefore, researchers need to explore the mechanism of Hh signaling pathway in more depth and find new targets for tumor-targeted therapy and inflammatory injury repair.
Figure 1. Classical canonical and proposed non-canonical Hedgehog signaling
Canonical Hh signaling pathway
The key ligands of the canonical Hh signaling pathway are Shh, Ihh, and Dhh. By autocrine, paracrine, and endocrine means, Shh binds to the receptor transmembrane protein-patch-1 (Ptch1) of the Hh signaling pathway. Thereby, the inhibition of smoothing (Smo) by Ptch1 under normal conditions is removed, and Smo can interact with related cytokines. Then, the Hh signaling pathway end effector glioma associated oncogene (Gli) family transcription factor nuclear translocation, regulation of Gli target gene expression, and finally the Hh signaling pathway is activated.
Ptch2 shares 54% homology with Ptch1, but tissue expression and its role in signaling pathways are significantly different from Ptch1. Ptch2 is highly expressed in seminoma and helps mediate Dhh activity in stem cell development. Studies have also shown that Ptch2 has a reduced inhibitory activity against Smo in the absence of binding to the receptor. In vertebrates, the Gli family contains three transcription factors, Gli1, Gli2, and Gli3. Gli1 is the only full-length transcriptional activator, and Gli2 and Gli3 play a positive or negative regulatory role in post-transcriptional and post-translational modification. In the case where Shh binds to the receptor, Gli2 aggregates in the primary cilia and activates transcriptional activity to counteract the negative regulation of Gli3. In the absence of ligand, the Hh signaling pathway original-suppressor of fused (SUFU) has a negative regulation of the Hh signaling pathway. It is anchored in the cytoplasm by binding directly to the Gli transcription factor, thereby preventing Gli from activating the target gene. At the same time, SUFU forms an inhibitory complex and interacts with DNA-bound Gli1, thereby inhibiting Gli1-induced gene expression. In addition to being regulated by SUFU, Gli1 can also be regulated by Dyrk1 kinase. Dyrk1 kinase enhances Gli1 activity through phosphorylation of multiple serine/threonine sites, which in turn induces Gli1 aggregation in the nucleus and Gli1-mediated transcription. Gli transcription factors can activate a variety of target genes, including Hh signaling pathway feedback factors (such as Gli1, Ptch1), proliferation-related factors (such as Cyclin-D1, MYC), apoptosis-related factors (such as Bcl-2, caspase-2), angiogenesis-related factors (such as ANG1/2), epithelial-mesenchymal transition-related factors (such as SNAIL), and stem cell self-renewal related factors (such as NANOG, SOX2).
Non-canonical Hh signaling pathway
In addition to the classical signal pathway axis, there are also non-canonical pathways associated with the Hh signaling pathway. There are two possibilities for this signaling pathway: one is the activation of the signal pathway initiated by Ptch1/Smo but without the participation of Gli transcription factors; the other is the activation of Gli transcription factors without the involvement of Shh or Ptch1/Smo. Studies have shown that multiple signaling pathways can directly induce enhanced Gli activity, such as K-Ras, TGF-β, PI3K-AKT, and PKC-α. In particular, K-Ras, which appears to independently induce Gli1 activation in the absence of the Shh signaling pathway, does not affect K-Ras-induced Gli1 activation in the case of SUFU knockout. In addition, Gli protein can be negatively regulated by p53, PKA, and PKC-δ. Overexpression of P53 attenuates the transcriptional activity of Gli, while knockdown of the P53 gene enhances its activity. In addition, the P53 gene also interacts with TAF9 to inhibit the transcriptional activity of Gli. PKA is specific for the regulation of Gli1, which directly phosphorylates the 374th threonine site of Gli1, promotes cytoplasmic localization of Gli1 and attenuates Gli1 activity.
Figure 2. Inhibition of components of the Shh Pathway in cancer
Hh signaling pathway and pancreatic cancer
Abnormal activation of the Hedgehog signaling pathway is closely related to the development of pancreatic cancer and the maintenance of malignant biological characteristics. Gli-1 is a transcriptional regulator of the end of the signal pathway, which transmits the extracellular Hedgehog signal into the nucleus, thereby initiating transcription of the target gene and thus plays an important role in the Hedgehog signal transduction pathway. During embryonic development, the Hedgehog signal transduction pathway regulates the normal development of the pancreas and is not expressed or only slightly expressed in the normal mature pancreatic tissue. Liu et al. showed that Gli-1 gene silencing alone is ineffective in the process of epithelial-mesenchymal changes. However, pancreatic cancer cells play an important role in the inhibition of epithelial-mesenchymal changes mediated by transforming growth factor β1 and epidermal growth factor. Zhao et al. believe that the cyclopamine nuclear cross-linked polymer micelle system blocks the expression of Gli-1 and improves the radiotherapy effect of Cs-137. The cyclopamine nuclear cross-linked polymer micellar system combined with radiotherapy is an effective treatment for pancreatic cancer and tumor-associated stroma.
Hedgehog signaling pathway and liver cancer
The Hedgehog signaling pathway is closely related to the activation of hepatic stellate cells. As the major nuclear transcription factor of the Hedgehog pathway, Gli-1 plays an important role in the activation of hepatic stellate cells. The aberrantly activated Hedgehog signaling pathway may activate its downstream gene Snail by enhancing Gli-2 activity. Therefore, the liver cancer cells have the ability to metastasize and invade, easily spread or metastasize to surrounding tissues, and promote the occurrence and development of hepatocellular carcinoma. After 15 days of implantation of mouse liver cancer ML-1 cells in B6 mice, liver cancer model mice were treated with Smo inhibitor GDC-0449 (vismodegib). It was found that after treatment with GDC-0449, the size of the tumor and the degree of infiltration of liver cancer cells were significantly reduced. The expression of Shh pathway gene also changed (including up-regulation of Shh expression and down-regulation of Smo expression), indicating that GDC-0449 can effectively alleviate liver cancer. Glypican 3 as a biomarker of hepatoma cells and a mediator of the Hedgehog pathway is associated with Hedgehog pathway-mediated survival of hepatic stellate cells. This suggests that it may play an important role in the development of liver cancer.
Hedgehog signaling pathway and basal cell carcinoma
Mutations in Ptch-1 in the Hedgehog pathway play an important role in the development of sputum-like basal cell carcinoma, manifested in its developmental defects and tumorigenicity. Stimulation and nutrient deprivation of the Hedgehog pathway synergistically activates the Hedgehog pathway in fibrosis of the squamous cell carcinoma cell, which is also associated with increased levels of transcription factors for Hedgehog pathway-associated genes such as the Wnt signaling gene. Ozgur et al. found that inhibition of the Hedgehog signaling pathway has a significant clinical response to basal cell carcinoma and sputum-like basal cell carcinoma syndrome. It can also rule out the eyelid contents of some patients, and its drug-related adverse reactions are relatively easy to control in most cases. Ching et al. demonstrated that even if Hedgehog pathway inhibitors are not effective treatments, they can alleviate the adverse effects of surgery and increase their efficacy. Therefore, patients with basal cell carcinoma can consider a combination of treatment options. The study found that vismodegib has a positive effect on local late-stage cancer and metastatic cancer of basal cell carcinoma; studies by Brinkhuizen et al. have also confirmed this view. At the same time, Hedgehog pathway inhibitors significantly reduce basal cell carcinoma of the skin.
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