Figures
Schematic representation of Notch canonical and non-canonical pathway. Initiation of Notch signaling occurs on ligand binding to the receptor. There are five typical Notch ligands Delta like, 1, 3, and 4 and Jagged 1 and 2 with a Delta-Serrate-Lag 2 (DSL) domain, while atypical ligands include DNER, F3/Contactin and NB-3 without a DSL domain and four Notch receptors Notch 1–4.58 Proteolytic cleavage by disintegrin and metalloproteases family proteases (MMP), and gamma-secretase releases active Notch intracellular domain (NICD).59 The biological process is mediated by Notch via canonical and noncanonical pathways. In the canonical Notch pathway, NICD translocates to the nucleus and binds to the transcription factor CSL. Mastermind co-activators activate the CSL-NICD complex leading to suppression of differentiation and maintenance of stemness by activating transcriptional targets HES1 and HEY1. HES1 increases the stemness-related genes in CRC cells and CSC surface markers CD133, ALDH1, and ABCG2. In noncanonical pathway, atypical ligand interacts with receptor promoting differentiation by formation of CSL-NICD-Deltex complex.56,57,60.
The process of Hedgehog pathway begins on binding of specific ligands such as exo-secretion ligand Shh bind to the trans-membrane receptor PATCHED1 (PTCH1) it initiates the HH-GLI pathway. Binding of this ligand to the receptor inhibits SMOOTHENED (SMO) protein leading to its translocation to primary villi of the cell and inhibition of GLI repressors (mostly GLI3R) and activation of intracellular signaling cascade. The cascade causes translocation and nuclear activation of the transcription factor Gli2. In parallel to this, the transcription factor Gli2 is upregulated playing an important role in cell proliferation, regulation and cell fate determination by turning on specific gene expression. It also causes transcription of three GLI zinc finger transactivation factors PTCH1, GLI1, HIP. Targeted inhibition of SMO and GLI1 could lead to cell death. Therefore, directly or indirectly inhibiting HH-GLI pathway could potentially eradicate the tumor and CSC population in the tumor.58,63.
Wnt signaling pathway with and without ligand binding and APC mutations. β-catenin is a protein kept under low cytoplasmic concentration by the destruction complex mainly regulating the Wnt pathway. The destruction complex consists of the tumor suppressor protein adenomatous polyposis coli (APC); casein kinase 1 (CK1) and glycogen synthase kinase 3b (GSK3-b); and Axin2, which scaffold the complex together. The membrane receptor complex is formed by frizzled (Fzd) and low-density lipoprotein receptor–related protein 5/6 (LRP5/6). In the absence of Wnt ligands, this membrane receptor complex is not engaged. Thereafter, CK1 and GSK3-b phosphorylate β-catenin at specific serine and threonine residues and leads to priming of its recognition by the U3 ubiquitin ligase β-transducin repeat-containing protein (β-TRCP). As a consequence, β-catenin is ubiquitinated and targeted for proteosomal degradation.67 Gene transcription is actively repressed in the nucleus as TCF transcription factors are bound to corepressor (Groucho).67 In the presence of Wnt ligands, they bind to Fzd and LRP5/6 coreceptors and trigger the formation of Dvl-Fzd complex. Also, it leads to phosphorylation of LRP by GSK3-b. The destruction complex is dissolved as this phosphorylation recruits the scaffolding protein Axin2 to the coreceptors.68 As a result, β-catenin stabilization occurs and can therefore accumulate in the cytosol. Subsequently, β-catenin translocates in the nucleus where it converts TCF into a transcriptional activator. This step is mediated by the displacement of the Groucho protein and recruitment of coactivators that include CBP, BCL9, and PYG.69 This recruitment ensures efficient transcription of genes that are important regulators of stem cell fate (LGR5, ASCL2), cell proliferation (C-MYC), and also, negative regulators of the pathway (Axin2). In CRC, truncating mutations in APC are frequently observed. In such mutations, there is inefficient targeting of β-catenin for degradation as the destruction complex is not properly formed. Therefore, even in the absence of external signal, β-catenin can accumulate and form active transcription factor complexes with TCF proteins in the nucleus.70.
Hippo Pathway for tumor suppression. Inactivation of YAP/TAZ leads to oncogenic transcriptional module. Its inactivation is due to activation of hippo kinases MST1/2 that facilitates activation of LATS1/2 thereby phosporylating and retaining YAP/TAZ in the cytoplasm via 14–3-3 or being subjected to proteasomal or autophagy-induced degradation. Followed by this, suppression of TEAD-mediated gene transcription occurs. On the other hand, inactivation of hippo kinases occurs due to myriad reasons. Inactivation of hippo kinases leads to dephosphorylation of YAP/TAZ and translocates inside the nucleus inducing TEAD target gene expression. However, recent studies highlight Hippo-YAP-independent activation of TEAD too.77,78.
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