5.1.1

Extracellular Mechanical Cues and YAP1/TAZ Activities

Mechanotransduction systems are important signals for growth and cell proliferation. Every cell responds to mechanical signals from the environment. Changes in elasticity/stiffness of the extracellular matrix (ECM) or force of traction/compression from adjacent cells regulates YAP1/TAZ subcellular localization. These mechanical forces are continuously transmitted among cell-cell or cell-ECM contact, which allows the rapid adjustment of cell stiffness via actomycin cytoskeleton contraction, cell shape and organ size control. Regulation of YAP1/TAZ via mechanical cues can be independent of Hippo signaling relying on Rho-GTPases and F-actin or the focal adhesion kinase (FAK)-Src-phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) pathway. However, a cell detachment mediated apoptosis termed anoikis is dependent on Hippo signaling. Through cytoskeleton reorganization, detachment leads to increase in LATS1/2 kinase and inactivation of YAP1 and TAZ. Anoikis is thought to be the major pathway by which differentiated intestine epithelial cells (IECs) undergo apoptotic cell death as they are sloughed off when the cell reaches the villi tips. Currently, it is not clear what role Hippo signaling plays in normal physiological regulation of anoikis in the intestine; however, in cancer cells, knockdown of YAP1 and TAZ restores anoikis-mediated cell death. In addition, cell polarity also regulates Hippo signaling. Crumbs is a mechanosensor that regulates cell polarity and was first discovered in D. melanogaster. Increase in Crumbs protein leads to increase in the apical surface, and YAP1/TAZ is phosphorylated via canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade, which is initiated by integrins and Src family kinases.

5.1.2

Cell Density and YAP1/TAZ Activities

Hippo pathway is highly active via cell-cell contact and is essential for contact growth inhibition. High cell density activates LATS kinase and leads to YAP1 inactivation, whereas low cell density inactivates LATS and results in YAP1 activation. Contact growth inhibition can be reversed via YAP1 overexpression, providing further evidence for the importance of Hippo signaling in regulating tissue and cell proliferation.

5.1.3

Stress and YAP1/TAZ Activities

Several stress signals regulate YAP1/TAZ activity, including chemical, heat, hypoxia, energy stress, endoplasmic reticulum (ER) stress, and oxidative stress. Heat shock or high concentration of sodium arsenite, a chemical stressor to induce heat shock proteins, activates MST1/2 and results in YAP1/TAZ cytoplasm localization. Hypoxia, on the other hand, modulates Hippo signaling through E3 ubiquitin ligase SIAH2-mediated degradation of LATS2, resulting in the activation of YAP1/TAZ to promote tumor cell proliferation and growth. Glucose deprivation rapidly activates LATS1/2 to phosphorylate YAP1/TAZ. In addition, low cellular glucose levels can activate AMP-activated protein kinase (AMPK), which can directly phosphorylate YAP1 and inhibit YAP1/TAZ/TEAD target gene transcription. Studies in Drosophila show that mTORC1 and mTORC2 induces Yki nuclear localization, while decreased mTORC1/2 prevents Yki target gene activation. In eukaryotic cells, the endoplasmic reticulum (ER) is the primary subcellular organelle that is important for protein folding, lipid and sterol biosynthesis, and calcium storage. ER stress occurs when unfolded or misfolded proteins accumulate in the ER lumen. ER stress activates unfolded protein response (UPR) to reduce unfolded/misfolded protein through ER membrane expansion, protein synthesis reduction, and quality control. YAP1 is important for ER expansion and UPR activity via PKR-like ER kinase (PERK)—eukaryotic initiation factor 2α (eIF2α) axis to reduce ER stress. Oxidative stress is associated with increased production of reactive oxygen species. Forkhead box protein O1 (FoxO1) regulates antioxidant genes, including catalase and manganese superoxide dismutase (MnSOD), to exert protective effect against oxidative stress. YAP1 forms a complex with FoxO1 on the promoters of the MnSOD antioxidant genes and stimulates their transcription, while inactivation of YAP1 suppresses FoxO1 activity and decreases antioxidant gene expression, implicating the role of YAP1 in modulating the FoxO1-mediated antioxidant response.

5.1.4

Growth Factor Pathways (EGF) and YAP1/TAZ Activities

The family of epidermal growth factor (EGF) ligands activates YAP1/TAZ through LATS-dependent and -independent manner. EGF activates PI3K dissociating 3-phosphoinositide-dependent protein kinase 1 (PDPK1) from LATS1/2-MST1/2-SAV1 complex, and leading to LATS1/2 phosphorylation and activation of YAP1/TAZ. In addition, EGFR-Ras-MAPK inhibits LATS1/2 activity by promoting LATS binding to AJUBA proteins (Ajuba LIM protein). The Hippo-independent pathway is through an EGFR-mediated cleavage of the ERRB4 receptor intracellular domain, resulting in YAP1 binding and nuclear translocation.

5.1.5

G-Protein-Coupled Receptors (GPCRs) and YAP1/TAZ Activities

YAP1/TAZ nuclear activities are regulated by the GPCRs. Studies have shown that several ligands, such as bioactive lipids, lysophosphatidic acid (LPA), and sphingosine-1-phosphate (S1P), inhibit Hippo kinase and activate YAP1 and TAZ through their GPCRs LPA receptor (LPAR) and S1P receptor (S1PR), respectively. Whereas YAP1/TAZ activity is inhibited by protein kinase A (PKA)-mediated LATS1/2 kinase activation upon ligand epinephrine and GαS-coupled GPCRs binding. In addition, GPCRs are the most diverse group of the plasma membrane receptors, which regulate the actions of hundreds of extracellular molecules. Therefore, the YAP1/TAZ regulation by GPCRs suggests that the Hippo pathway is modulated by a large number of signals yet to be identified.

5.3.1

Wnt Signaling

The Wnt signaling pathway is a well-studied pathway that is involved in maintaining normal intestinal epithelial homeostasis. β-Catenin plays a central role in canonical Wnt signaling pathway. In the absence of Wnt ligands, the destruction complex including adenomatous polyposis coli (APC), axin, casein kinase Iα (CKIα), and Glycogen synthase kinase 3β (GSK3β) serves as a negative regulator of β-catenin. The complex phosphorylates β-catenin and retains it in the cytoplasm, where it is ubiquitinated and degraded by the proteasome. Upon canonical Wnt ligands (Wnt1, Wnt3a, and Wnt8) binding to Wnt receptors frizzled and single-membrane-spanning low-density receptor-related protein (Lrp) 5/6 receptor, β-catenin is stabilized and translocated into the nucleus, where it interacts with T-cell factor (TCF) and lymphoid enhancer factor (LEF) transcriptional factors to activate the transcription of its target genes. Well-controlled Wnt signaling is essential for IEC proliferation, survival, polarity, cell fate determination. Inhibition of Wnt/β-catenin signaling results in rapid loss of ISCs and transient-amplifying cells and crypt structures . Mutation or dysfunction of the destruction complex leads to β-catenin stabilization and induces tumorigenesis. The Apc gene is the most frequently mutated gene in colon cancer. In addition to the canonical Wnt pathway described above, the noncanonical Wnt signaling pathways include Wnt polarity, Wnt-Ca ^{2 +} , and Wnt-atypical protein kinase C pathways and is independent of β-catenin. The noncanonical Wnt ligands include Wnt4, Wnt5a, and Wnt11, which bind to receptor frizzled as well as tyrosine-protein kinase transmembrane receptor ROR2 and receptor tyrosine kinase (RYK). The noncanonical Wnt signaling pathway activates c-Jun N-terminal kinases (JNK), profilin, Rho-associated kinase (ROCK), calcineurin/Ca ^{2 +} /calmodulin-dependent protein kinase II (CaMKII), and contributes to the developmental processes such as planar cell polarity and epithelial cell migration. ROR2 or Wnt5a knockout mice showed similar phenotypes including perinatal lethal, dwarfism, facial abnormalities, and shortened limb. In addition, the deletion of ROR2 exhibited defects of cell orientation and cell polarity.

YAP1 and TAZ are highly integrated to both the canonical and noncanonical Wnt signaling pathways. YAP1 overexpression or MST1/2 deletion modulate nuclear β-catenin and downstream target genes. However, studies have also shown that YAP1 and TAZ cytoplasmic protein expression restricts Wnt signaling by limiting the activity of a positive Wnt signaling regulator Dishevelled (DVL) in ISCs or nuclear translocation of β-catenin. Moreover, this is a bidirectional cross talk as YAP1 and TAZ activity are activated by the Wnt pathway. YAP1 and TAZ are part of the β-catenin degradation complex and upon canonical Wnt activation, YAP1 and TAZ nuclear localization are increased and are major effectors of the Wnt response. However, recent work suggests that Wnt-induced YAP1 and TAZ do not share a common mechanism of activation as β-catenin. Alternative Wnt signaling regulates LATS1/2 leading to enhanced YAP1 and TAZ activation ( Fig. 5.2 ).

Cross talk between YAP1/TAZ and Wnt signaling pathways. Several opposing views and different mechanistic regulation of YAP1/TAZ and Wnt signaling are proposed. (A) YAP1/TAZ cytoplasmic expression represses β-catenin activity by limiting a positive Wnt signaling regulator called Dishevelled (DVL). (B) The β-catenin degradation complex (APC, Axin, CKIα, and GSK3β) includes YAP1. The complex phosphorylates and retains β-catenin in the cytoplasm, where it is ubiquitinated and degraded by the proteasome. Upon canonical Wnt ligand (Wnt1, Wnt3a, and Wnt8) binding to frizzled and single-membrane-spanning low-density receptor-related protein (Lrp) 5/6 receptor, β-catenin, and YAP1/TAZ translocate into the nucleus, leading to activation of β-catenin and YAP1/TAZ targets genes by binding to transcriptional activators T-cell factor (TCF) and lymphoid enhancer factor (LEF) or TEAD, respectively. (C) APC can directly restrict YAP1/TAZ by regulating upstream Hippo kinases. This restriction is independent of Wnt/β-catenin signaling, and deletion or mutation of APC, which is often observed in colon cancer, leads to inhibition of Hippo kinases and activation of YAP1 activity. (D) The noncanonical Wnt ligand Wnt5a/b binds to receptor frizzled as well as tyrosine-protein kinase transmembrane receptor ROR2 leading to activation of the G-protein-coupled receptor through Gα12/13. Subsequent activation of Rho GTPases (RHO) inhibits LATS1/2, which in turn activates YAP1/TAZ activity.

5.3.2

Notch Pathway

The Notch pathway is a highly conserved pathway that is essential for cell fate determination and maintenance of ISCs. Notch signaling is a juxtacrine signaling pathway where the signal sending cells contains membrane-bound ligands (type I transmembrane proteins of the Delta/Serrate/Lag-2 (DSL) family) and activates receiving cell through binding to cell surface Notch receptors (Notch1-4). Notch receptor activation leads to the cleavage of the Notch intracellular domain (NICD), which is then translocated to the nucleus. NICD binds to the DNA-binding transcription factor CSL (CBF1/RBPjk/Su(h)/Lag1), leading to the increased transcriptional activity. Studies demonstrate that activation of Notch signaling in the intestinal epithelium increases the cell differentiation towards absorptive enterocytes, and inhibition leads to an increase in the secretory lineage (enteroendocrine, goblet, and Paneth cells). Notch inhibition leads to a significant decrease in active cycling ISCs. In addition, inhibition of Notch pathway has been considered as a promising target for cancer and increasingly reported in clinical trials in patients with solid tumor. The initial success of Notch inhibition in decreasing cancer growth was dampened by the major side effects including gastrointestinal toxicity and diarrhea. Interestingly, Hippo signaling component YAP1 physically interacts with NICD and modulates the Notch signaling outputs. ChIP-seq for the Notch DNA-binding mediator Rbp-J revealed the direct binding of Rbp-J to the YAP1 and Tead2 loci. Several Notch pathway members such as Notch1, Notch2, Sox9, and Hes1 are upregulated with YAP1 overexpression in the hepatocyte. Notch2 was identified as a direct YAP1/TAZ/TEAD target gene. In addition, overexpression of NICD leads to increased expression of YAP1 and Tead2, suggesting a positive feedback loop between Hippo and Notch signaling pathways ( Fig. 5.3 ).

Cross talk of YAP1/TAZ, Notch, BMP, and Hedgehog pathways. A positive feedback loop between Hippo and Notch signaling pathway. YAP1 physically interacts with NICD and modulates the Notch signaling outputs. Several Notch pathway members are upregulated with YAP1 overexpression. In addition, overexpression of NICD leads to increased expression of YAP1/TAZ. YAP1 forms a complex with SMAD1 and promotes the BMP downstream targets. YAP1 binds to and negatively regulates the activity of GLI transcription factors, which, in turn, represses Hedgehog target genes. GLI , glioma-associated oncogene homolog; NICD , notch intracellular domain.

5.3.3

Bone Morphogenetic Protein (BMP)

Bone morphogenetic protein (BMP) is part of the transforming growth factor-β (TGF-β) super family protein and specifically binds to BMP receptors (type I and type II BMP receptor). BMPs are precursor protein with N-terminal signal peptide and C-terminal mature peptide. In the intestine, BMP2 and BMP4 are expressed in the mesenchyme and their receptors are expressed in the epithelium. BMPs are a negative regulator of ISC self-renewal and proliferation and crucial for the intestinal development and intestinal epithelial tissue homeostasis. The canonical BMP signaling cascade is initiated by binding to serine/threonine kinase receptors and forming a heterotetrameric complex. Subsequently, the constitutively active type II receptor phosphorylates downstream receptor-regulated SMAD (R-SMAD; SMAD1, 5, and 8) leading to heterodimerization with SMAD4 to regulate downstream target genes. Recent studies have revealed a cross talk between Hippo and BMP signaling pathways. TAZ induces BMP4 transcription, which enhances TAZ downstream targets by promoting SMAD1/5 intracellular signaling. YAP1is also involved in physically forming a complex with SMAD1, which promotes downstream targets of BMP ( Fig. 5.3 ).

5.3.4

The Hedgehog (Hh) Signaling Pathway

The Hedgehog (Hh) signaling pathway is conserved signaling pathway that is essential for tissue maintenance, renewal, and regeneration and therefore crucial for normal intestinal homeostasis. The Hh signaling pathway involves Hh ligands (Sonic Hedgehog, Indian Hedgehog, and Desert Hedgehog), receptors (e.g., Patched-PTCH1, GAS1, CDON, and BOC) as well as signaling cascade substrates that include smoothened (SMO), suppressor-of-fused (SUFU), glioma-associated oncogene homolog (GLI), and the downstream targets. The Hh pathway is initiated by binding of the ligands to its canonical receptor protein patched homolog 1 (PTCH1), and coreceptors growth arrest- specific gene 1 (GAS1), cell adhesion molecule-related down- regulated by oncogenes (CDON), and brother of CDON (BOC), resulting in the release of the SMO suppression facilitating its entry onto the primary cilia. SMO then promotes the disassociation of the SUFU/GLI complex, resulting in nuclear translocation and activation of the Hh pathway transcriptional effectors GLI proteins. YAP1 binds to and negatively regulates the activity of GLI transcription factors, which, in turn, represses Hh pathway target genes. However, it has been reported that Hh signaling can increase YAP1 by increasing its protein levels. This suggest that Hh and Hippo signaling form a negative feedback loop ( Fig. 5.3 ).