**5. Canonical Hedgehog pathway**

The canonical Hedgehog pathway is characteristically over active in many forms of metastatic breast cancer and is associated with enhanced migration, invasion, stemness and self-renewal of cancer cells [69–72]. Over activity of the Hedgehog pathway is also associated with playing a role in promoting EMT [71]. The Hedgehog ligand was first discovered in the *Drosophila* fruit fly [73]. Several human Hedgehog ligands have also been identified that are involved in cell growth and controlled organ formation by insuring that the tissue reaches the accurate size and position. In the adult, this pathway normally remains quiescent. However, activation of the Hedgehog pathway may be triggered during tissue maintenance and regeneration [74]. A link between Hedgehog signaling and developmental defects was first discovered in 1996, and later that year a link between Hedgehog signaling and cancer was found when the tumor suppressor gene patched (PTCH) was discovered [67]. Soon afterwards, the Hedgehog cell service signaling transducer, smoothened (Smo), was discovered and found to have the potential to function as an oncogene. These findings lead to the development of the Hedgehog pathway inhibitor, cyclopamine, and successful clinical trials using cyclopamine and similar agents followed [75]. Hedgehog ligands are produced by three different genes. The first gene is the Indian Hedgehog (Ihh) and is found in gut, skeletal muscle, and chondrocytes [76]. The second gene is the Desert Hedgehog (Dhh) and is expressed in the testis [76].

The third gene is the Sonic Hedgehog (Shh) and is involved in many developmental processes [76]. Shh is called a morphogen since the signal of this ligand relies on its concentration [77]. The Shh is produced from zone of polarizing activity (ZPA), which is located on the posterior side of the limb bud in the embryo [78]. The Hedgehog pathway has a link with the formation of specific types of humans cancer [74]. After the transcriptional and translational process occur, this ligand is secreted as a precursor protein, and the ligand is subsequently subjected to several post translational modifications [73]. Autocatalytic cleavage then splits the ligand into two parts. One part is the signaling molecule, while the other part appears to have no function. A cholesterol molecule and palmitic acid moiety are then added to C-terminal and N-terminal of the signaling piece, leading to an increase in its hydrophobicity, localization and binding to the receptor [73]. The canonical Hedgehog signaling pathway has several vital components which play a role in modulating signal intensity. Most of the components within the Hedgehog pathway include the Hedgehog ligand, the PTCH receptor, Smo, and the cytosolic complex and downstream effectors, which consist of suppressor of fused (SUFU) and Gli family of proteins. The Gli family is an important component of the Hedgehog pathway which is divided into three forms known as Gli1, Gli2, and Gli3. Gli transcription factors can activate the signal, have dual function to stimulate or impede the signal [79]. A number of kinases, such as GSK3β, CK1α, and protein kinase A, are known to be essential in the regulation of Hedgehog signaling [80]. The PTCH receptor of this pathway is located in the lipid raft microdomains of the plasma membrane [8].

Activation of the Hedgehog pathway can be blocked in the absence of ligand expression or a lack of mutation in PTCH and/or Smo [79]. In such cases, the inhibitory effect of PTCH on Smo is intact and Hedgehog ligand transport to the cell membrane is prevented and receptor activation and signal transduction does not occur [79]. In contrast, activation of the Hedgehog pathway will result in conditions when the Hedgehog ligand is highly expressed, and/or when mutation of PTCH and/or Smo occurs [79]. In these conditions, inhibitory effect of PTCH on Smo is absence and Smo can freely travel to the cell membrane, leading to the phosphorylation of SUFU and the transcription factor in the cytosolic complex. Once this occurs, Gli separates from the cytosolic complex proteins and then translocates into the nucleus where it promotes an increase in the Hedgehog target gene expression [79]. Recent studies have shown a direct connection between EMT and stemness of breast cancer resulting that is directly associated with the activation of the canonical Hedgehog signaling and the development of tumor recurrence and metastasis [71].

**Figure 4B** shows the effects of γ-tocotrienol treatment on signaling protein levels and activation within the Hedgehog pathways. Results show that the Hedgehog Shh ligand is relatively high in MDA-MB-231 breast cancer cells in the vehicle-treated control group. Similarly, PTCH2 receptor, Smo, GSK3β, and Gli1 were highly expressed, while the inhibitor for Hedgehog signaling SUFU displayed a relatively low level of expression in the vehicle-treated MDA-MB-231 human breast cancer cells (**Figure 4B**). Treatment with γ-tocotrienol induced a dose-dependent decrease in Shh ligand expression, as well as a dose-responsive reduction in PTCH2 receptor, Smo, GSK3β, and Gli1, and a corresponding increase in SUFU protein levels, as compared to MDA-MB-231 cells in the vehicletreated control group (**Figure 4B**). These data indicated that γ-tocotrienol inhibition of EMT is also mediated by a suppression of canonical Hedgehog pathway and provides further evidence that γ-tocotrienol treatment may provide significant benefit in the treatment of metastatic breast cancer.
