Thesis
Reference
Origins, Regulation and Function of the Hippo pathway components in Hydra
AL HADDAD, Sarah
Abstract
The hippo pathway is a tumor-suppressor signaling cascade that regulates cellular mechanisms such as proliferation, cell death, cell differentiation and cell migration to ensure a proper development and regeneration of organs. Its function is well studied in Drosophila and vertebrates however, nothing is known about its activity in pre-bilaterians. Hydra belongs to Cnidaria, a sister group of Bilateria, and represents high regenerative capacity. The goal of this work is to study the relevance of the Hippo pathway during homeostasis and regeneration in Hydra. First, we present a wide phylogenetic analysis of 46 gene families related to the pathway. Next, we study the function of the pathway by knocking down several key players during homeostasis and regeneration conditions. Furthermore, we monitor the effect of mechanical tension on the activity of the pathway through its downstream effector YAP.
AL HADDAD, Sarah. Origins, Regulation and Function of the Hippo pathway components in Hydra. Thèse de doctorat : Univ. Genève, 2018, no. Sc. Vie 6
URN : urn:nbn:ch:unige-1154098
DOI : 10.13097/archive-ouverte/unige:115409
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES
Département de Génétique & Évolution Professeure Brigitte GALLIOT
Origins, regulation and function of the Hippo pathway components in Hydra
THÈSE
présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biosciences
moléculaires
par
Sarah Al Haddad de
Beyrouth (Liban)
Thèse n° Genève
Atelier d’impression REPROMAIL 2018
Acknowledgements
I would like to start by thanking my Ph.D. advisor Brigitte Galliot who accepted me into her lab, and tried to push me to develop my scientific and personal skills. She taught me two important lessons in life, (1) no matter what your status is, you should always remain human and generous, and (2) whatever achievements you reach in life, you can always achieve more with the right decisions and investments.
I would also like to thank Prof. Robbie Loewith and Dr. Charisios Tsiairis for their interest in my work and for accepting to be a part of my PhD thesis committee.
A big thanks to all the lab members: Szymon Tomczyk for his great friendship, Chrystelle Perruchoud for her big heart and dedication, Laura Iglesias for her trust and our funny discussions, Matthias Vogg for the time he spent correcting my thesis and for always laughing at my jokes, Quentin Schenkelaars for his fun spirit and joyful presence, Wanda Buzgariu for her honest care on all levels, Salima Boukerch for her generosity and yummy food, Nina Taciuc for always being there when needed, Denis Benoni for his technical help and inside jokes, Valérie Mino and Corinne Matthey for taking care of all the administrative work. I would also like to thank former lab members: Yvan Wenger for his help and contribution to my project, as well as Marie-Laure Curchod and Kazadi Ekundayo.
This Ph.D. has not only brought me scientific and personal development. I also got to meet an amazing partner and colleague who have made my journey so exciting, filled with love, support, and so much fun! Nenad Suknovic, thank you for all the efforts you have put to make it easier, especially at the end. Hayete enta, moje dušo!
I am very grateful to my family for always being there, no matter the day or the time, and for teaching me how to take control over stressful situations. Papa, thank you for your wisdom and our priceless and supportive conversations. Maman thank you for all the sacrifices you’ve made so I can go on with my studies. To Sylvain, thank you for our eternal complicity and trust, for being able to understand our mutual PhD struggles. To Etienne, my partner in brainstorming, your smart view of life taught me a lot and saved me from many dead-end situations. Bhebkon!!
To Téta Nounou, Zeina, Kimi, Nastia, Pascale, Atef, Riad and Yulia, thank you for your continuous and honest support. Forever grateful.
I am very thankful to have met amazing friends during my studies. Thank you Samar for ongoing support and great memories, Asli for a rare and precious friendship, Rita for being my home far away from home, Carlos for providing me with so much inspiration and motivation, Kamila and Adrien members of the amazing “6” club, Piango, Djordje, Baba, Yogesh, Nuno, Claudio, Melissa, François for the motivated rugby team! To all my Lebanese friends in Lebanon or abroad: Samar, Aline M., Eliane, Charbel, Jizel J, Aline Z.,
Table of Contents
Acknowledgements ... 5
Résumé... 9
Abstract...11
List of figures ... 13
List of tables ... 13
List of abbreviations ... 14
Introduction- Hippo pathway 1. Origin and Discovery of the Hippo pathway ... 17
2. Components of the Hippo pathway ... 18
Core pathway in mammals ... 19
Downstream effectors: YAP/TAZ and the transcriptions factors ... 20
3. Regulation of the Hippo pathway ... 21
3. 1 Apical-basal polarity complexes ... 21
Ø The Par/α-PKC complex ... 22
Ø The Scrib complex ... 23
Ø NF2-FRMD6-Kibra complex ... 23
Ø The Crumbs complex ... 23
Ø Other complexes and independent regulators ... 24
3. 2. Planar-cell polarity complexes ... 26
3. 3. The Wnt pathway ... 27
3. 4. G-protein-coupled receptor signaling ... 30
3. 5. Mechanical cues ... 31
Ø Cell adhesion and geometry ... 32
Ø Matrix stiffness ... 32
Ø Cell density ... 33
3. 6. Stress signals ... 34
4. Role of the Hippo pathway ... 36
4. 1. Hippo pathway in embryonic development ... 36
4. 2. Hippo pathway in organ development and homeostasis ... 36
4. 3. Hippo pathway in regeneration ... 39
Regeneration in mammals ... 39
Regeneration in non-mammalian organisms ... 41
2. Developmental processes in Hydra ... 49
3. Hydra in homeostasis and regeneration ... 51
3. 1. Homeostasis ... 51
3.2. Regeneration ... 52
AIM of the project...53
Results Chapter 1: Successive evolutionary waves built an almost complete Hippo pathway in epithelial cells of eumetazoan ancestors...55
Chapter 2: Role of the Hippo pathway in Hydra 1. Role of the Hippo pathway during homeostasis in Hydra ... 173
2. The Hippo pathway is required for head but not for foot regeneration in Hydra ... 176
3. hyYAP is a sensor of mechanical forces in vitro ... 179
Discussion 1. Challenges and limitations of the project ... 183
2. Terminally differentiated regions exhibit a high expression of the Hippo pathway components in Hydra ... 185
3. Dominant epithelial expression of the Hippo components reflects a strong transcriptional regulation in slow cycling stem cells ... 186
4. YAP is required to regulate cell death during homeostasis in Hydra ... 187
5. The Hippo pathway regulates the injury-induced cell death dependent regeneration in Hydra ... 189
6. hyYAP is a sensor of mechanical forces in mammalian cells ... 191
Conclusion and perspectives ...195
References ...197
Résumé
La voie Hippo est une cascade de signalisation cellulaire dont la fonction principale est la suppression de tumeurs par la régulation de mécanismes cellulaires tels que la prolifération, la mort cellulaire, la différentiation, et la migration. Cette régulation assure également le bon développement et la régénération des organes. Bien que de nombreuses recherches sont menées sur le rôle de la voie Hippo chez la Drosophile et les vertébrés, rien n’est actuellement connu sur son rôle chez les espèces non- bilateriennes malgré leur capacité de régénération élevée. Parmi ces espèces, l’hydre appartient à la famille des cnidaires, qui constituent le groupe-frère des bilateriens.
Après bissection de l’animal à n’importe quel niveau du tronc, l’hydre est capable de régénérer les parties manquantes à partir des deux fragments obtenus. Le but de la présente recherche est d’étudier l’expression des composants de la voie Hippo chez l’hydre, ainsi que son activité et sa régulation dans le contexte de l’homéostasie et de la régénération.
Ce travail présente une étude phylogénétique détaillée de 48 familles de gènes qui constituent la partie fondamentale de la voie Hippo, les régulateurs qui fonctionnent en amont de la partie fondamentale, les effecteurs en aval de la cascade, et les gènes cibles.
Nous avons ainsi recherché et étudié ces composants dans 26 espèces appartenant à différents groupes phylogénétiques afin d’émettre des hypothèses sur leur émergence et leur conservation. Nous avons également analysé l’expression de ces gènes chez l’hydre en fonction des différentes parties du tronc et des trois populations de cellules souches.
Il s’avère que la majorité des gènes impliqués dans la régulation et la partie fondamentale sont fortement exprimés dans les parties différentiées c’est-à-dire aux extrémités de l’animal, ainsi que dans les cellules souches qui prolifèrent lentement (i.e.
les cellules souches épithéliales). Par contre, les gènes cibles putatifs sont exprimés d’une façon dominante dans la partie proliférative du corps et dans les cellules souches qui cyclent rapidement (i.e. cellules souches interstitielles).
Ensuite, nous avons étudié la fonction de la voie Hippo pendant l’homéostasie et la régénération de l’hydre en réduisant l’activité de quelques protéines dont l’importance pour la voie Hippo est majeure. Nos résultats montrent que YAP est nécessaire pour garder un équilibre homéostatique en régulant le niveau de mort cellulaire dans l’animal. De même, pendant la régénération, l’activité de LATS, YAP, TEAD et TAO contribue à la formation de la nouvelle tête mais pas du pied. Par contre, l’inhibition de ces gènes n’empêche pas la régénération de la tête mais cause un retard dans l’apparition des premiers signes de régénération.
De plus, nous avons testé l’effet de la régulation mécanique sur la localisation intracellulaire de hyYAP et son activité en fonction de la densité cellulaire, comme cela a
Pour conclure, ce travail montre que (A) la voie Hippo est conservée chez l’hydre et que son émergence est antérieure aux bilatériens, (B) hyYAP est essentiel à l’homéostasie de l’hydre en régulant le niveau de mort cellulaire, (C) la voie Hippo influence la cinétique de régénération de la tête mais pas celle du pieds, et finalement (D) hyYAP est sensible aux modulations de la tension mécanique sur les cellules.
Abstract
The hippo pathway is a tumor-suppressor signaling cascade that regulates cellular mechanisms such as proliferation, cell death, cell differentiation and cell migration to ensure a proper development and regeneration of organs. Its function is well studied in Drosophila and vertebrates however, nothing is known about its activity in pre- bilaterians, despite the high regeneration capacities that these phyla possess. Hydra belongs to Cnidaria, a sister group of Bilateria, that emerged at the base of eumetazoans and represents high regenerative capacity as bisection of the animal at any level of the body leads to a complete regeneration of the missing parts. Our main goal was to study the relevance of the Hippo pathway during homeostasis and regeneration in Hydra.
In this work, we present a wide phylogenetic analysis of 48 gene families from core components, downstream effectors, target genes and upstream regulators of the Hippo pathway, by studying their emergence and conservation across evolution in 26 species.
Next, we analyze the spatial and cell-type related expression of these genes in Hydra and depict a different regulation in the three stem cell populations and along the animal, where most of the core and upstream regulators are highly expressed at the differentiated extremities of the animal and in slow cycling cells, whereas the putative target genes are highly expressed in the proliferative zone of the animal and in fast cycling cells.
Next, we study the function of the pathway during homeostasis and regeneration by knocking down key players of the pathway. Our results show that the downstream effector YAP is crucial to maintain homeostasis in Hydra by keeping a low cell death level in the animal. Similarly during regeneration, the activity of LATS, YAP, TEAD and TAO is important to complete head regeneration but not foot regeneration. Surprisingly, the knockdown of these genes did not inhibit head regeneration as expected, but delayed its process.
Furthermore, we study the effect of mechanical regulation on hyYAP in human cells, since huYAP is well known to sense cell density and act accordingly. Our data shows that hyYAP senses cell density and localizes to the cytoplasm when cells are dense and tension is low, and translocates to the nucleus when cells are sparse and tension is high.
In the nucleus, hyYAP is active and promotes transcription of genes through its interaction with TEAD.
In conclusion, this work shows that (A) the Hippo pathway is very well conserved in Hydra and emerged long before bilaterians, (B) hyYAP is essential to maintain
List of figures (Introduction and Chapter 2)
Figure 1: Timeline of the discovery of the Hippo pathway genes Figure 2: Components of the canonical Hippo pathway in mammals
Figure 3: The apical basal complexes in the regulation of the Hippo pathway in mammals
Figure 4: The crosstalk between YAP and the Wnt pathway.
Figure 5: Mechanical forces regulate YAP activity via the F-actin network Figure 6: YAP activity throughout development and adulthood in mice Figure 7: Histology and cytology in Hydra
Figure 8: Developmental processes in adult Hydra
Figure 9: YAP is important for Hydra fitness in intact animals Figure 10: YAP regulates cell death in Hydra
Figure 11: HR is delayed with the knockdown of the Hippo pathway Figure 12: The Hippo pathway regulates head but not foot regeneration
Figure 13: hyYAP changes its sub-cellular localization according to cell density Figure 14: The activity of hyYAP depends on cell density in vitro
Figure 15: hyYAP interacts with hyTEAD and huTEAD
Figure 16: Summary of the function of YAP in Hydra and vertebrates
List of tables (Introduction and Chapter 2)
Table 1: Summary of the Hippo pathway mutations and their effect in different model organisms during regeneration.
List of abbreviations
• AJ: Adherens Junction
• AMOT: Angiomotin
• ANKRD: Ankyrin repeat domain
• APC: Apical Basal Complex
• BrdU: Bromodeoxyuridine
• Cas-3: Caspase-3
• CCNE: CyclinE
• cDNA: complementary DNA
• Cdx2: Caudal-type homeobox protein 2
• CIP: contact inhibition of proliferation
• co-IP: co-Immunoprecipitation
• CSC: Cancer Stem Cells
• CTGF: Connective tissue growth factor
• Cyr61: Cysteine-rich angiogenic inducer 61
• Ds: Dachsous
• DSS: dextran sodium sulfate
• ECM: Extracellular matrix
• eESC: ectodermal epithelial stem cells
• EMT: Epithelial to mesenchymal transition
• EP: electroporation
• Ex: Expanded
• Fj: Four-jointed
• FoxO1: Forkhead box protein O1
• FR: foot regeneration
• Fzi: Frizzled
• gESC: gastrodermal epithelial stem cells
• HEK293T: human embryonic kidney 293 T antigen
• hpa: hours post-amputation
• hpd: hours post-dissociation
• hpt: hours post- transfection
• HR: head regeneration
• HU: Hydroxy-urea
• huTEAD: human-TEAD
• huYAP: human-YAP
• Hv: Hydra vulgaris
• hyTEAD: Hydra-TEAD
• hyYAP: Hydra-YAP
• ICM: Inner Cellular Mass
• IF: Immunofluorescence
• In: Input
• ISC: Interstitial stem cells
• LATS: Large tumor suppressor
• LD: Low density
• mCherry: monomer Cherry
• MEC: myo-epithelial cells
• MST: Mammalian STE20-like protein kinase
• NF2: Neurofibromin-2
• PatJ: Pals1-associated tight junction protein
• PCP: Planar Cell Polarity
• Pitx2: Pituitary homeobox 2
• PKC: Protein kinase C
• RNA: ribonucleic acid
• RNA-Seq: RNA-sequencing
• S75A: Serine 75 to Alanine mutation
• TAZ: Tafazzin
• TBS8x: Tead-binding-sites 8x
• TCF: Transcription factor
• TE: Trophectoderm
• TEAD: TEA domain
• TJ: Tight Junction
• TSG: Tumor Suppressor genes
• U-2 OS: Bone osteosarcoma
• Vgll4: Vestigial-like 4
• WCE: Whole cell extract
• YAP: yes-associated-protein
• Yki: Yorkie
• ZO-1/ZO-2: zona occludens
I. Introduction to the Hippo pathway
1. Origin and Discovery of the Hippo pathway
Since the early 90’s, many fields of research were directed towards understanding the origin and cause of cancer, to produce drugs that might cure this invasive, and very often, lethal disease. Some sets of genes cause tumor initiation and progression and others prevent its formation. These two sets were named oncogenes and tumor suppressor genes (TSGs), respectively. In 1953, Carl Nordling suggested that carcinogenesis is not due to a single mutation but to an accumulation of mutations that lead to tumor formation. Based on this hypothesis, Alfred Knudson established in 1971 his hypothesis best known as the “two-hit hypothesis” (Knudson, 1971) where he did statistical analysis on inherited retinoblastoma and observed that some people inherit one mutated version of the retinoblastoma gene and another normal version; this is the first hit. However, this mutation is not effective unless the normal allele undergoes a mutation and therefore, both alleles are not functional. This two-hit hypothesis applies to TSGs but not to oncogenes. In the case of oncogenes, a mutation of one allele of a given gene is enough to initiate over-proliferation, implying that the mutation of oncogenes is dominant whereas TSG mutation is recessive. Therefore, in order to be effective, TSGs must follow the two-hit rule with mutations on both alleles to delete their activity.
Following these discoveries, genetic screens in human cancer cells and Drosophila were established to identify TSGs. In 1994, Watson and colleagues from the University of California performed loss of function mutations and identified the first 50 tumor suppressor genes in Drosophila, e.g. Lgl, Expanded, Fat, Notch (Watson et al., 1994). In 1995, a series of genetic screens in Drosophila identified WARTS, the ortholog of LATS in vertebrates and a component of the Hippo pathway, as a tumor suppressor whose
Figure 1: Timeline of the discovery of the Hippo pathway genes
The discovery of the Hippo pathway components began with Warts/Lats in Drosophila imaginal discs in 1995, and took about 12 years to identify the rest of the genes and to establish the Hippo pathway as known today.
2. Components of the Hippo pathway
The Hippo pathway consists of a kinase cascade, transcriptional co-activators and transcription factors. Its activation and repression takes place in the cytoplasm and directly inhibits the activity of its downstream effectors, preventing them from trans- locating to the nucleus and promoting proliferation. After deciphering its role in Drosophila, the Hippo pathway was also studied in mammals where orthologs were found. However in mammals, some upstream regulations show a higher complexity and a slight divergence from the Drosophila pathway, without altering its main growth- regulatory role.
Core pathway in mammals
In mammals as in Drosophila (mammals/Drosophila), the kinase cascade starts with phosphorylation of Ste20-like kinases MST1-2/Hippo, which in turn phosphorylate the Large Tumor Suppressor LATS1-2/WARTS kinase, along with their adaptor protein SAV1/Salvador and kinase-activator MOB1A-B/MATS, respectively (Hansen et al., 2015a; Yu et al., 2015). Once activated, LATS1-2/WARTS will phosphorylate the co- activator YAP/Yki on the S127 residue, preventing its translocation to the nucleus and retention in the cytoplasm by 14-3-3 regulatory proteins (Fig. 2). However, upon phosphorylation on the S381 residue, YAP undergoes proteasomal degradation. When the kinases are inactive, YAP/Yki translocates to the nucleus and binds to the TEAD/Scalloped family of transcription factors to promote the transcription of proliferation and anti-apoptotic genes (Pan, 2010; Piccolo et al., 2014a).
Figure 2: Components of the canonical Hippo pathway in mammals
Scheme showing the core Hippo pathway in the cytoplasm, and the downstream effectors in the nucleus. The MST kinase is auto-phosphorylated and is activated by SAV. MST phosphorylates the kinase LATS, which in turn is activated by MOB. Activation of LATS promotes YAP phosphorylation on S127 and retention in the cytoplasm by 14-3-3 regulatory protein. When the kinase cascade is inactive YAP translocates to the nucleus and binds to its transcription factor
Downstream effectors: YAP/TAZ and the transcriptions factors
YAP is an oncogene that plays a key role in cell proliferation. Its overexpression can lead to over-proliferation and tumor formation, leading to the reprogramming of cancer cells into cancer stem cells (Gregorieff et al., 2015). In mammals, YAP has a paralog named TAZ that is similarly regulated, with minor changes in the number of serine/threonine phosphorylation sites, i.e. YAP has five and TAZ four (Piccolo et al., 2014a). Two out of these five residues are very important for the phosphorylation and inactivation of YAP i.e. S127 (S89 for TAZ) and S381 (S311 for TAZ) (Zhao et al., 2010a). YAP is a very stable protein although it gets degraded upon S381 phosphorylation, whereas TAZ has a half- life of less than 2 hours, making its degradation the way for its inhibition (Zhao et al., 2010b). One of the best readouts of the pathway activity is the YAP/TAZ sub-cellular localization, however YAP can act independently of the Hippo pathway via several upstream regulators.
The TEAD family represents four main transcription factors (TEAD1-4) that interact with YAP when the Hippo pathway is inactive. A double knockout of TEAD1 and TEAD2 in mice resembles the phenotype of YAP knockout, i.e. a decrease in cell proliferation and an increase in cell death (Sawada et al., 2008; Zhao et al., 2008). The target genes that result from the YAP/TEAD interaction in vertebrates are TEAD transcription factors, CTGF, Cyr61, ANKRD, CCNE, and others (LaQuaglia et al., 2016). It was recently shown that YAP can act as a tumor suppressor by binding to other transcription factors such as p73 to induce apoptosis in response to DNA damage. Furthermore, the transcription factor RUNX3 interacts with YAP and TEAD forming a ternary transcriptionally inactive complex. In growth stimulation contexts, RUNX3 separates from the complex and YAP/TEAD promote proliferation. However, in growth inhibition context, TEAD separates from the ternary complex, creating a phospho-YAP/RUNX3 interaction. This shows that the activity of YAP depends on its DNA-binding partners, and is regulated by cellular mechanisms (Kim et al., 2018).
3. Regulation of the Hippo pathway
The Hippo pathway is regulated by a series of upstream pathways that activate or inhibit its core components, to maintain a homeostatic proliferation rate and to prevent the overgrowth of organs or tumor formation. These upstream regulators can be molecular pathways (Vogel and Sheetz, 2006) such as apical-basal polarity complexes, planar-cell polarity (PCP) complexes, GPCR signaling, but also mechanical cues that act through cytoskeleton remodeling thus affecting cell dynamics and fate (Yu and Guan, 2013).
3. 1 Apical-basal polarity complexes
The apical-basal polarity of epithelial cells is defined by cellular junctions, where cells can adhere or communicate together through tight junctions (TJ) (also known as septate junctions in invertebrates), adherens junctions (AJ) and desmosomes (or intermediate filaments). These junctions involve a large number of polarity complexes that determine their position and boundaries along the apical to distal axis. TJs are present at the apical part of the cell linking epithelial cells together to form a tight and partially permeable wall. They control the paracellular permeability of the cells throughout the epithelium, as well as the intramembrane diffusion (Shin et al., 2006). AJs are present at the basolateral part and connect actin bundles between two cells. Both TJs and AJs divide the plasma membrane into an apical part and a basolateral part respectively, which confers the apical-basal polarity of the epithelial cells. Interestingly, many of the molecular pathways and protein complexes located at the junction level, act upstream of the Hippo pathway (Fig. 3).
Figure 3: The apical basal complexes in the regulation of the Hippo pathway in mammals
The different complexes acting upstream of the pathway are localized at the tight junctions (a- PKC, NF2/FRMD6/Kibra, Crumbs) or at the adherens junctions (a-catenin, SCRIB). The orange frame of proteins implies their phosphorylation.
Ø The Par/α-PKC complex
The Par apical complex is formed by four proteins, partitioning-defective 3 (PAR3) also known as Bazooka (Baz) in vertebrates, partitioning-defective 6 (PAR6), cdc42 (small GTPases) and atypical protein-kinase (α-PKC), and decides the boundary of TJs in the cell. The α-PKC complex inhibits the activity of the Hippo pathway resulting in the
Ø The Scrib complex
The Scrib complex works as a neoplastic tumor suppressor and a module that prevents the apical complexes to re-localize at the basal pole of the epithelial cells. It is localized at the baso-lateral plasma membrane domain and is formed by Scribble (SCRIB), Discs large (DLG) and Lethal (2) giant-larvae (LGL). In Drosophila, it localizes at the SJs whereas in mammals it localizes at the AJs. Scribble acts as an adaptor for the core cassette where MST and Lats are assembled into a complex, to promote Lats activation and TAZ inhibition. In breast cancer stem cells, when SCRIB is delocalized from the plasma membrane, the Hippo pathway is inactive and the high activity of TAZ leads to cancer stem cell formation (Cordenonsi et al., 2011). On the other hand, the Scrib complex antagonizes the activity of the PKC complex by inhibiting PKC activity, leading to Hippo pathway activation and YAP inhibition (Yu and Guan, 2013).
Ø NF2-FRMD6-Kibra complex
NF2/Merlin and FRMD6/Expanded (Ex) are tumor suppressors that belong to the FERM (Ezrin-Radixin-Myosin) cytoskeleton-membrane linker protein family. They localize at the TJs through interaction with AMOT, Pals1 and PatJ, providing junction stability (Martin-Belmonte and Perez-Moreno, 2011). Merlin is encoded by the NF2 locus (neurofibromatosis type 2) and together with FRMD6, they regulate proliferation and differentiation (McCartney et al., 2000). Kibra is a protein that physically interacts with NF2 and FRMD6, forming a complex at the apical part of polarized epithelial cells that recruits Lats to the apical pole, facilitating its phosphorylation and activation by MST. In some cases, Merlin can translocate to the nucleus to inhibit the CRL4 ubiquitin ligase, leading to an active nuclear Lats that will impair the activity of YAP and its dependent transcription (McCartney et al., 2000). A knockdown of Merlin leads to tumor formation and metastasis in mice (McClatchey et al., 1998).
Ø The Crumbs complex
The Crumbs complex is assembled on the apical pole of epithelial cells and contains the
whereas its overexpression leads to inactivation of the Hippo pathway and increase in Yki activity, suggesting that the amount of Crumbs is critical for the function of the Hippo pathway (Ling et al., 2010; Robinson et al., 2010). In mammalian cells, the knockdown of Crumbs 3 results in YAP nuclear translocation and activation (Varelas et al., 2010).
Ø Other complexes and independent regulators
Angiomotin (AMOTs) are localized to TJs and interact extensively with apical complexes to maintain the stability of TJs. AMOT binds the WW domains of YAP/TAZ through its PPxY motif and promote its tight junction or actin cytoskeleton localization, thus preventing its nuclear translocation independently of phosphorylation (Zhao et al., 2011). AMOT can also inhibit YAP/TAZ activity through Lats activation and YAP/TAZ phosphorylation. On the other hand, an interaction between AMOT and F-actin is mediated through Lats phosphorylation, which may suggest that the loss of Lats phosphorylation leads to F-actin/AMOT disruption and AMOT/YAP interaction to prevent YAP from nuclear translocation and activation (Piccolo et al., 2014a). AMOT is a clear example about the independent regulation of YAP/TAZ, outside of the traditional Hippo cascade.
α-catenin is a junctional protein and a tumor suppressor that acts as a linker between the actin cytoskeleton and the trans-membrane E-cadherin at the level of AJs. In keratinocytes, α-catenin interacts with YAP via 14-3-3 protein and inhibits its activity. In this case, unlike AMOT, YAP needs to be phosphorylated on the S127 residue to induce 14-3-3 binding, followed by α-catenin binding. This complex formation leads to YAP sequestration to AJs, far from the nucleus where it might be reactivated (Schlegelmilch et al., 2011). In α-catenin mutants, mice skin develops hyper-proliferation and leads to a similar phenotype in YAP-
Many other junctional regulators are shown to regulate the activity of the Hippo pathway or to bypass the cascade and affect YAP/TAZ function, independently of the pathway. E-cadherin, Ajuba and Liver kinase B1 (LKB1) inhibit YAP’s activity by promoting its phosphorylation. At the TJs, the zona occludens proteins ZO-1 and ZO-2 repress TAZ activity and activate YAP, respectively (Yu and Guan, 2013). The protein tyrosine phosphatase kinase (PTPN14) regulates the Hippo pathway by directly interacting with YAP, which leads to its cytoplasmic retention and decreased activity (Liu et al., 2013).
In summary, the apical-basal polarity seems to be a very important regulator of the Hippo pathway. This can occur by the sequestration of the Hippo pathway kinases to the apical pole of the cell to promote their phosphorylation or by recruiting YAP/TAZ to TJs and AJs. In both cases, YAP/TAZ activity is inhibited.
3. 2. Planar-cell polarity complexes
Planar cell polarity (PCP) is the collective alignment of apico-basal polarized cells across a tissue plane (Devenport, 2014). As mentioned before, epithelial cells are oriented by their apical-basal polarity and they follow this axis for orientation. The planar cell polarity is a perpendicular axis to that of apico-basal polarity and helps clustering epithelial cells into one aligned and well-oriented layer (Devenport, 2014). However, PCP is not restricted to epithelial cells, it is also important in mesenchymal cells to help their intercalation and migration. PCP behavior is controlled by two systems: the Frizzled (Fzi) system and the Fat/Dachsous (Ds) system (Simons and Mlodzik, 2008).
Fat and Ds are two atypical cadherins that interact together by forming intercellular heterodimers through the phospho-mediation of four jointed (Fj). In Drosophila, Fat acts as a tumor suppressor that inhibits the activity of Yki by activating Expanded and Warts.
Ds and Fj are usually expressed in an opposite gradient manner; this gradient is very important for Fat activation (Feng and Irvine, 2007). For a given level of Ds, Fat activity is inhibited which leads to the apical localization of atypical myosin Dachs. The polarization of Dachs promotes Zyxin-Warts interaction, which leads to Warts degradation and Yki activation (Rauskolb et al., 2011).
The casein-kinase 1 (CK1) homologue Dco (discs overgrown) enhances Fat activity by phosphorylating its intra-cellular domain, whereas the palmitoyltransferase Approximated (App) promotes Dachs apical translocation by relieving Fat inhibition.
Another modulator of the Fat pathway is Lowfat (Lft) that promotes Hippo pathway activation by increasing the stability of Fat and Ds (Mao et al., 2009; Matakatsu and Blair, 2008; Sopko et al., 2009).
In mammals, there are four Fat orthologs (Fat 1-4) and two Ds orthologs. Fat4 is the vertebrate homologue that corresponds to Drosophila Fat, and its knockout in mice does not show any effect on the activity of Lats or YAP (Mao et al., 2011). On the other hand, mammalian cells lack a well-conserved Dachs, which suggests that the function of the mammalian Fat pathway may not be conserved upstream of the Hippo pathway in mammals.
3. 3. The Wnt pathway
The Wnt pathway is an important signaling cascade in development and morphogenesis.
It is involved in cell proliferation, cell migration, cell differentiation and regeneration (MacDonald et al., 2009). The core of Wnt pathway revolves around β-Catenin activity and its shuttling from the cytoplasm to the nucleus. When the Wnt pathway is off, a destruction complex is formed in the cytoplasm and is composed of Axin, GSK3, and APC.
This complex leads to β-Catenin phosphorylation by GSK3 and its degradation after ubiquitination with β-TrCP. When Wnt binds to its ligand Frizzled (Frz), the destruction complex is disrupted and β-Catenin accumulates and translocates to the nucleus to bind to its transcription factor TCF, and promote expression of Wnt target genes (Clevers, 2006).
Recent studies have shown that the Hippo pathway and the Wnt pathway crosstalk in the cytoplasm and YAP/TAZ constitute an important part of the Wnt destruction complex. YAP/TAZ bind to Axin and recruit the β-TrCP for ubiquitination of phospho-β- Catenin. This means that YAP/TAZ are inactive and sequestered in the cytoplasm when Wnt is OFF (Azzolin et al., 2014). In this case, depletion of YAP/TAZ leads to transcription of β-Catenin/TCF target genes in mouse ES cells, highlighting the importance of YAP/TAZ in β-Catenin regulation. Further more, the overexpression of transcriptionally defective and cytoplasmic YAP leads to crypt degeneration in mouse intestine due to β-Catenin inactivation (Barry et al., 2013). When Wnt is ON, Axin releases YAP/TAZ from the complex leading to β-Catenin accumulation and YAP/TAZ translocation to the nucleus to activate Wnt-dependent target genes (Fig. 4).
In both cases, it seems that in the context of Wnt pathway, YAP/TAZ work in favor of each scenario: in the nucleus, when Wnt is ON, YAP/TAZ promote Wnt/β-Catenin mediated transcription, and when Wnt is OFF, YAP/TAZ act as activators of β-Catenin degradation and inhibitors of its nuclear entry. It is also important to mention that the degradation of β-Catenin is also true for TAZ whose degradation is not phospho- dependent as YAP (Azzolin et al., 2012). This could also mean that Wnt activation stabilizes TAZ expression, whereas in the context of “Wnt OFF”, YAP promotes to the
So what happens when the Hippo pathway is ON? In this case, YAP/TAZ will be automatically retained in the cytoplasm, in their transcriptionally inactive state, which can lead to their recruitment by the destruction complex and β-Catenin degradation.
This indirectly suggests that an active Hippo pathway inactivates the Wnt pathway whereas an inactive Hippo pathway will lead to the activation of Wnt/ β-Catenin signaling and Wnt/YAP/TAZ signaling. This also means that the inactivation of Hippo pathway is sufficient to promote β-Catenin activation, regardless of Wnt as in cardiomyocytes where the knockout of MST1/2, Lats2 and SAV leads to β-Catenin activation and over proliferation (Heallen et al., 2011a). In mice liver, knockout of MST1/2 leads to hepatocellular carcinoma in correlation with YAP/TAZ, Wnt/β-Catenin, and Notch signaling activation (Kim et al., 2017). The activation of both Wnt/β-Catenin and Wnt/YAP/TAZ pathways is not always required and is context dependent; in intestinal crypts, the knockdown of YAP/TAZ does not affect the homeostatic function of the intestine whereas the knockdown of LGR4/5 Wnt receptor leads to degeneration of the crypts, both in vivo and in vitro (de Lau et al., 2011). On the other hand, YAP/TAZ are required for regeneration of organoid crypts in culture, and for crypt growth in vivo when Wnt is activated (Azzolin et al., 2014). Some of the Wnt target genes are regulated by YAP/TAZ and β-Catenin such as Sox2 and Snai2 in cardiomyocytes, and Birc5 and Bcl2l2 in colorectal cancer (Piccolo et al., 2014a). Altogether, these results suggest an intimate yet complicated regulation between YAP/TAZ and Wnt signaling, and suggest that the Wnt pathway and Hippo pathway act antagonistically to maintain homeostasis and avoid tumor formation.
Figure 4: The crosstalk between YAP and the Wnt pathway.
When Wnt pathway is off, the destruction complex retains YAP and β-catenin in the cytoplasm, whereas its activation leads to YAP and β-catenin nuclear translocation and expression of target genes. Orange frame implies that proteins are phosphorylated.
3. 4. G-protein-coupled receptor signaling
G-protein-coupled receptors (GPCRs) are a family of protein receptors on the surface of the cell that are coupled to small heterotrimeric G proteins. These receptors recognize extracellular signals such as hormones, nutrients and other secreted proteins and, depending on the G protein coupled to each receptor, these extracellular signals are transmitted to the appropriate downstream effectors (Lappano and Maggiolini, 2011). It was expected that growth factors and hormones could regulate the activity of the Hippo pathway until it was demonstrated that the diffusive molecules SIP (sphingosine-1- phosphate) and LPA (lysophosphatidic acid) stabilize and activate the YAP/TAZ. Gα12/13
and Gαq/11 proteins coupled to their receptors activate the Rho-GTPases. This activation leads to F-actin stabilization through the activation of the ROCK/LIM/COFILIN and PKA pathway, which in turn inactivates Lats1/2 promoting YAP/TAZ activation and nuclear translocation, with the expression of target genes i.e. CTGF, CYR61 and ANKRD1 (Yu et al., 2012). In contrast, signals mediated through Gαs coupled receptors after binding to glucagon or epinephrine lead to Lats activation and YAP/TAZ cytoplasmic retention and inhibition. This shows that YAP/TAZ activation in the context of GPCRs highly depends on the activated receptor (Yu et al., 2013).
In uveal melanoma, a mutation in Gαq/11 causes a hyper-proliferation due to YAP constant activation. Verteporfin is a drug that inhibits the interaction between YAP and TEAD, which leads to transcriptional arrest of target genes. When the function of YAP is disrupted by verteporfin in uveal melanoma, the growth of tumor cells is arrested.
Another study showed that the overexpression of Gαq leads to YAP nuclear translocation by inhibiting the activity of Lats or the activity of AMOT independently of Lats to activate YAP (Yu et al., 2014; Yu et al., 2012). Other ligands that act through GPCR signaling are the Wnt proteins and more specifically Wnt5a/b and Wnt3a. These proteins activate the non-canonical Hippo pathway by binding to the F-GPCRs family (Frizzled) and activating the Gα12/13,which in turn activate Rho-GTPases and inhibit Lats1/2. This leads to YAP activation and TEAD-mediated transcription (Park et al., 2015).
3. 5. Mechanical cues
At every moment of the day, our cells undergo mechanical forces from their surrounding environment that affect their shape and activity, namely shear stress from the movement of blood and fluids along a cell boundary, heart pumping, pressure, gravity, and skeletal generated forces (Vogel and Sheetz, 2006). Our tissue architecture is very complex yet robust, and such complexity inhibits cells to over-proliferate in physiological conditions. These external forces applied on the cells via the extra-cellular matrix (ECM) and the tension caused by other cells, is counterbalanced by internal forces that keep the shape of the cytoskeleton organized and functional, and confer the 3D structure of our cells. When these forces are disturbed by unusual mechanical strains, cell signaling is perturbed causing tissue malfunction and diseases (Vogel and Sheetz, 2006). It was already known that cell shape and cell junctions control proliferation, apoptosis and stem cell differentiation. However, the link between this physical and biochemical property of a cell remained a mystery for a long time.
In 2011, the piccolo lab showed that stretching a single cell plated on ECM-like substrate allows F-actin polymerization and YAP/TAZ activation followed by increased cell proliferation and inhibition of differentiation (Dupont et al., 2011). Later, several studies established YAP/TAZ as mechanotransduction proteins, acting in a Hippo pathway independent context. However, the interpretation of these mechanical signals is not straightforward and depends on each cell type and the nature of the external signal (Codelia and Irvine, 2012). These signals are mechanical stimuli linked to the cell- adhesive area and cell geometry, the stiffness of the cell substrate, cell density, and shear stress caused by blood and liquid flow (Fig. 5).
Figure 5: Mechanical forces regulate YAP activity via the F-actin network
YAP is a sensor of mechanical forces and its activity is monitored by high and low mechanical tensions on the cell membrane and in the cytoskeleton. These forces can change according to cell stretching, matrix stiffness, cell density, and the accumulation of stress fibers. With high mechanical tension, YAP is nuclear and promotes proliferation whereas with low mechanical forces, YAP is inactive and retained in the cytoplasm.
Ø Cell adhesion and geometry
When cells are exposed to small adhesive area, they are not well attached and their shape is rounded and very small. This leads to YAP/TAZ cytoplasmic retention and inactivation. When a cell is attached on a large adhesive area, high tension is applied on its membrane leading to the activation of YAP/TAZ in the nucleus (Aragona et al., 2013).
The knockdown of YAP/TAZ causes cell detachment and induced anoikis, a form of programmed cell death that occurs when cells are detached from ECM (Zhao et al., 2012). However, this does not apply on cancer cells where the Hippo pathway is not activated, leading to migration of mutated cells and YAP-mediated over-proliferation and metastasis.
forces, which causes the YAP/TAZ nuclear translocation and activation. However, when ECM is soft and cells are exposed to less than 1.5 kPa, YAP/TAZ are cytoplasmic and inactive and cell proliferation is impaired (Aragona et al., 2013). Stretching a monolayer of epithelial cells also leads to YAP/TAZ activation. The knockdown of Lats on soft substrate does not lead to YAP/TAZ activation suggesting that Lats does not play a role in this context and that YAP/TAZ regulation is Lats independent. However, few studies showed that the inhibitors of Lats, LPA and S1P, lead to Rho mediated YAP/TAZ activation by inhibiting Lats and that YAP activation depends on Lats inhibition in a JNK dependent context (Codelia et al., 2014; Wada et al., 2011; Zhao et al., 2012).
Ø Cell density
Contact inhibition of proliferation (CIP) describes the ability of cells to sense their surroundings and stop proliferating once they reach a confluence that occupies all the cultured space. In cancer, this sensing ability is lost leading to over-proliferation and tumor formation (Sharif and Wellstein, 2015). In high cell density conditions, YAP is phosphorylated at S127 and retained in the cytoplasm whereas in low cell density conditions, YAP is nuclear and active (Zhao et al., 2007). However, a high cell density is hard to define because cells do not stop proliferation when they undergo cell-cell contact. There should be a threshold that sets parameters to stop proliferation. Indeed, cell density affects cell proliferation in two steps. In the first step, cells enter in contact and activate the E-cadherin/α-catenin system, which triggers Lats activation and a partial YAP inhibition for about 30%. In this case, 70% of the cells are still proliferating and this effect can be reversed by Lats knockdown. In the second step, cells continue to proliferate until they reach a very high confluence that triggers cell geometry change leading to small and rounded cells with smaller adhesion surface. At this stage, E- cadherin/α-catenin are not affected but the F-actin cytoskeleton undergoes remodeling via F-actin capping and severing proteins (CapZ, Cofilin and Gelsolin). These proteins establish a mechanical checkpoint by which cells are fully affected by mechanical strains (Aragona et al., 2013; Piccolo et al., 2014a).
adhesion components such as talins, vinculins, PTK2, and SRC kinases to promote F- actin polymerization and the formation of an actomyosin contractile network. Inhibition of muscle myosin and myosin light chain kinase by Blebbistatin and ML-7 respectively, leads to YAP/TAZ inhibition (Dupont et al., 2011; Wada et al., 2011). This cytoskeleton remodeling and actin dynamics is tightly regulated by the Rho pathway and inhibitors against the Rho-associated protein kinase (ROCK) such as Y27632 lead to YAP inactivation (Gaspar and Tapon, 2014). As mentioned earlier, this Rho-GTPases regulation can work independently or can be dependent of the Hippo pathway. It is clear that the activation step between F-actin fibers and YAP/TAZ is not well understood yet and might be cell type dependent. What is important to know is that YAP/TAZ regulation is not affected by the G-actin/F-actin ratio because in small and rounded cells, the total F-actin is higher than in spread cells and still YAP/TAZ are inactive (Connelly et al., 2010). YAP/TAZ mechanotransduction is inhibited very specifically by F-actin capping and severing, and the knockdown of one of the capping proteins Gelsolin, Cofilin or CapZ leads to YAP/TAZ nuclear translocation and transcription of target genes in mammals as well as in Drosophila (Aragona et al., 2013; Fernandez et al., 2011).
Recently, an interesting study was published about the independent role of the cytoskeleton in nuclear YAP translocation. In vitro, if a high force is applied directly on the nucleus, YAP bypasses the cytoskeleton regulation and enters the nucleus pores through active transport due to higher exposure of nuclear pores on the stretched nuclear membrane (Elosegui-Artola et al., 2017). Of course this does not apply in vivo by itself, however on a stiff substrate, cells perceive the high mechanical force from the ECM, which makes it impossible to bypass the actin-Rho pathway. In this case, the cytoplasmic and nuclear membranes are stretched, which may suggest that both cytoskeleton and nuclear remodeling are important for YAP nuclear translocation.
3. 6. Stress signals
Tissues and cells are often exposed to cellular and environmental stress from lack of
YAP/FOXO1 interaction and lead to apoptosis. On the contrary, the pathway is inhibited during Hypoxia, and YAP/TAZ are activated (Ma et al., 2015; Shao et al., 2014). During osmotic stress and high cell density, YAP activity is inhibited since TEAD localizes in the cytoplasm (Lin et al., 2017).
Glucose and amino acids are important regulators of metabolism. As it is known, the mTOR pathway regulates cell size and proliferation, in an amino acid-dependent manner and is activated by YAP/TAZ during nutrient shortage, by inducing the expression of LAT1, a target gene of YAP-TEAD mediated transcription (Hansen et al., 2015b). On the other hand, glucose deficit-generated stress activates Lats1/2 and inhibits AMPK, leading to YAP/TAZ inhibition. During cancer, cells follow the Warburg effect, which consists of using glycolysis as a main source of ATP and energy production. In this case, the activity of Lats1-chaperone Hsp90 is impaired by glycation and YAP/TAZ is activated, which indicates that glucose consumption enhances cell proliferation in cancer through YAP/TAZ (Park et al., 2018).
4. Role of the Hippo pathway
With all the complex upstream regulation mentioned above, downstream of the Hippo pathway lie a wide range of functions that affect cells during development, homeostasis, regeneration and cancer.
4. 1. Hippo pathway in embryonic development
During early stages of embryonic mice development, cell-fate decision happens during the pre-implantation stage when the inner cell mass (ICM) and trophectoderm (TE) are formed (Marikawa and Alarcon, 2009). TE is the outer cell layer in a blastocyst that gives rise to extra-embryonic tissue. These cells are well polarized and attached together via tight junctions (TJs), while expressing Cdx2, a marker of TE cells. ICM are non-polarized cells that form the inner mass of the blastocyst and highly express pluripotency markers, namely Oct4 and Sox2, leading to embryo tissue formation (Zernicka-Goetz et al., 2009). In these 2 layers, YAP/TAZ show a difference in cell localization at 16 cell-stage, where YAP/TAZ are nuclear in TE cells and cytoplasmic in ICM cells (Sasaki, 2015). The expression of Cdx2 in TE cells requires TEAD4-YAP mediated transcription. When TEAD4 is knocked down, mice embryos do not develop a proper TE layer, and all the blastocyst cells turn into ICM. Similarly, when Lats2 is overexpressed in TE cells, the expression of Cdx2 is suppressed. Furthermore, the knockdown of Lats1/2, AMOT or Merlin leads to a mass of TE cells and the tissues deriving from ICM are not developed (Nishioka et al., 2009). On the other hand, ICM contraction forces the outer layer of the blastocyst to stretch and cover the external surface of the embryo. This results in YAP nuclear localization and cell fate decision of TE. Furthermore, the inhibitory drugs Y27632 and Blebbistatin inhibit the activity of ROCK and non-muscle myosin II respectively, lead to YAP retention in the cytoplasm, and abolish the TE commitment (Kono et al., 2014). It is important to note that TAZ-KO mice can survive whereas YAP-KO mice die since blastomers cannot pass to morula stage. These findings suggest that YAP/TAZ are essential for early embryonic
proliferation and apoptosis in order to keep the homeostatic state of the tissue. The Hippo pathway regulates proliferation, apoptosis, differentiation and migration of cells.
In Drosophila for instance, the knockdown of Hippo, Warts, Ex, Kibra, or Merlin results in increased proliferation and tissue overgrowth in wings and eyes (Zhao et al., 2010a). A similar effect is observed with the overexpression of Yki (Yu et al., 2015). The effect of organ size regulation is conserved in mammals and more specifically in mice. Several studies were conducted in different organs with conditional knockouts targeting genes from the core, upstream regulators or YAP/TAZ.
In liver of adult mice, knockout of MST1/2, NF2 or Lats1/2 leads to liver overgrowth (Camargo et al., 2007). Yap overexpression in hepatocytes does not lead to over-proliferation, because upon YAP increase, apoptotic programs are initiated. In order to push the activity of Yap, a second signal provoked by injury or tissue damage is required. In response to this injury, Yap activates proliferation while inhibiting differentiation (Su et al., 2015). This YAP-induced overgrowth is reversible via apoptosis in over-proliferating cells, when YAP is back to its homeostatic level. However, YAP knockout in liver does not lead to organ size reduction as expected, but instead leads to bile duct defect. It was suggested that the reason behind this unchanged size is that TAZ overcomes the loss of YAP to maintain homeostasis (Yu et al., 2015).
Heart growth is determined in two phases; the first one at the embryonic level where cardiomyocytes proliferate to form a functional heart, and the second phase is post-natal where proliferation stops and the heart size is determined (Zhou et al., 2015). In mice heart, deletion of MST1/2, Lats1/2 and SAV in embryos, or the overexpression of YAP, leads to heart enlargement and cardiomyocytes hyperproliferation. Furthermore, conditional knockout of YAP leads to heart hypoplasia in a YAP-TEAD1 dependent manner. In postnatal phase, TAZ deletion had no effect on cardiomyocytes proliferation whereas a combined
Interestingly, in mice intestine, YAP/TAZ knockout has no effect on tissue homeostasis, whereas the overexpression of YAP leads to repression of Wnt signaling and proliferation of intestinal stem cells known as crypt cells. Knockout of MST1/2 and SAV results in decreased differentiation, crypt hyperplasia and intestinal adenoma (Barry et al., 2013; Cai et al., 2010).
In mice kidneys, the deletion of MST1/2 and SAV1 does not have any effect of kidney function. Curiously, the inactivation of TAZ and YAP does not have the same effect, as YAP is involved in nephron morphogenesis and TAZ deletion causes polycystic kidney formation (Reginensi et al., 2013). In zebrafish, YAP was also shown to be important for kidney development (He et al., 2015).
During mammary glands development, YAP does not affect the function of the glands, however during pregnancy, deletion of YAP leads to hyperplasia and overexpression of YAP abolishes terminal differentiation of the mammary cells (Chen et al., 2014).
A list of other organs in mice shows the implication of the Hippo pathway in their development and function such as the epidermis, lungs, pancreas, nervous system and muscles (Yu et al., 2015). One striking thing to conclude is that the dispensability of this pathway is context and organ dependent: the deletion of YAP can be insignificant as in intestine and mammary glands and the deletion of core genes such as MST1/2 in kidneys does not lead to overgrowth. But among these results, there is no clear evidence that YAP/TAZ actually regulates organ size, independently of the Hippo pathway because deletion of one or both does not lead to significant shrinking of the organs and reduction in size, it is rather that their conditional overexpression leads to significant expansion in some organs. Another observation concerns the function of YAP during homeostasis in certain cases: in a complete homeostatic state, YAP activity is very limited and its intervention is not required. However, during development or unusual
4. 3. Hippo pathway in regeneration
The Hippo pathway is implicated in regeneration response upon tissue damage in mammalian and other model systems. YAP activity is important for a complete regeneration in some organs such as limbs, liver, intestine, heart and others.
Regeneration in mammals
In liver of adult mice, YAP is expressed in the epithelium of bile duct. Upon inflammation or injury, YAP is activated and promotes proliferation of progenitors while reducing differentiation. In case of induced-bile duct obstruction, deletion of YAP causes inhibition of hepatocytes proliferation ad regeneration deficiency (Su et al., 2015).
In mice intestine, when the tissue is injured with exposure to dextran sodium sulfate (DSS), it develops acute colitis or inflammation of the inner lining of the intestine. YAP is highly upregulated in the crypts to ensure regeneration of the damaged part. If YAP is deleted, this leads to defective regeneration and loss of intestinal crypts (Cai et al., 2010). It is important to know that YAP/TAZ induce proliferation in the crypts when working with TEAD and induces differentiation when working with Klf4 in goblet (secretory) cells, which means that YAP activity does not always lead to cellular proliferation in the intestine and when regeneration is needed, YAP/TAZ work with TEAD to ensure the proper response (Imajo et al., 2015).
In the skin of mice, wound healing and cell renewal happen thanks to the stem cell basal layer, where YAP/TAZ are upregulated upon injury to promote wound healing. When YAP/TAZ expression is depleted, stem cells loose the ability of proliferation, and regeneration of the skin does not occur (Wang et al., 2017).
referred to as tissue repair rather than tissue regeneration (Wang et al., 2017).
The expression of YAP in the heart decreases with age, as the expression of its competitor VGLL4 increases. Upon myocardia injury, activated YAP rescues the function of the heart. Interestingly, when Mst2 is deleted, cardiomyocytes proliferation is not affected but the application of high stress reduces heart diseases (Del Re et al., 2014; Lin et al., 2014). Similarly, loss of Sav1 enhances cardiomyocytes proliferation and regeneration upon injury (Heallen et al., 2013).
It is important to know that YAP interacts with the transcription factors FoxO1 and Pitx2 to promote antioxidant gene expression. This may also help the heart to maintain its integrity (Shao et al., 2014).
In summary, this data suggests that the Hippo pathway regulates organ growth not only during development but its activity is also required for a complete regeneration and tissue repair. In postnatal periods, YAP activity appears at a very low level and is upregulated in case of injury or non-homeostatic state (Fig. 6). YAP/TAZ are considered as targets for regeneration medicine as they can dedifferentiate cells from pancreas and mammary glands back to their progenitor state, which leads to ex-vivo organoid formation followed by transplantation in vivo (Panciera et al., 2016).
Regeneration in non-mammalian organisms
In general, the regeneration ability varies between organisms and organs within the same species and decreases with the increase of organism complexity. For instance, regeneration in mammal limbs is restricted to wound healing whereas Xenopus limbs are fully regenerated by replacing the missing parts. Many non-mammalian organisms show a high dependency on the Hippo pathway and YAP to complete their regeneration.
In flatworms, also known as planarians, regeneration of full body after bisection occurs in a YAP-dependent manner as yki(RNAi) animals exhibit a delay in the proliferation wave at 48hpa, which leads to regeneration failure (Lin and Pearson, 2017). Similarly, during regeneration of imaginal discs in Drosophila, the activity of the Hippo pathway is repressed in cells close to the cut whereas yki is highly activated in cells at wound edges and neighboring to apoptotic cells (Grusche et al., 2011). In the midgut of adult Drosophila, the downregulation of yki abolishes proliferation after stress-induced infections in the intestine. In contrast, the downregulation of warts under the same stress conditions promotes intestinal stem cell proliferation but does not affect terminal differentiation (Grusche et al., 2011; Shaw et al., 2010). In Crickets, leg regeneration is regulated by the fat pathway and Merlin/Ex pathway as the knockdown of fat and dachsous abolishes proliferation in the regenerating axis resulting in regenerated shorter legs. However, when Merlin or expanded are silenced, cells over-proliferate resulting in longer legs (Bando et al., 2009). In Xenopus limbs and tail regeneration require YAP activation in the blastema, and does not occur upon YAP inhibition (Hayashi et al., 2014). In zebrafish, cell density differs along the blastema of regenerating fin i.e.
dense in the distal part and sparse in the proximal part. YAP sub-cellular localization and activity changes according to this cell density where it is mostly cytoplasmic in distal blastema and highly nuclear and active in proximal blastema. This regulation is tightly coupled to F-actin activity upstream of YAP and is disrupted upon YAP inactivation, indicating that YAP regulates fin regeneration under the control of mechanical forces and cytoskeleton signals (Mateus et al., 2015). These examples highly emphasize the important regenerative role of the Hippo pathway in bilaterian species
Species Mutation (lof, gof) Effect Liver Mouse YAP (lof) Inhibition of hepatocyte
proliferation and regeneration deficiency
Intestine Mouse YAP (lof) Defective regeneration and loss of intestinal crypts, or defective differentiation Skin Mouse YAP/TAZ (lof) Loss of proliferation,
regeneration inhibition
Heart Mouse - YAP (gof)
- MST (lof)
- SAV (lof)
- Rescues myocardia injury - Does not affect proliferation, reduces heart disease under high stress conditions - Higher proliferation of cardiomyocytes
Full body Flatworm yki (lof) Delay in the second proliferation wave and regeneration failure Imaginal
discs
Drosophila,
adult - Yki (lof) - Warts (lof)
- Abolishes proliferation in stem cells
-Enhances stem cell
proliferation, does no affect differentiation
Legs Cricket - Fat, Ds (lof) - Merlin, Ex (lof)
- Reduced proliferation, shorter regenerated legs - Over-proliferation, longer regenerated legs
Tail Xenopus,
tadpole YAP, TEAD (lof) Reduced proliferation, increased apoptosis, regeneration defect
Fin Zebrafish YAP (lof) Disruption of mechanical
regulation and inhibition of regeneration
Table 1: Summary of the Hippo pathway mutations and their effect in different model organisms during regeneration.
Loss of function (lof) or gains of function (gof) mutations affect the regeneration process in mammalian (mouse) and non-mammalian species (flatworm, Drosophila, cricket, Xenopus, and zebrafish).
4. 4. Hippo pathway in cancer
Cancer is one of the major causes of death in the world and the search for drugs that inhibit its spreading in the human body is restless. When talking about the Hippo pathway and the oncogenic role of YAP, it is expected that YAP/TAZ play an important role in cancer initiation and proliferation. YAP/TAZ induce the conversion of neoplastic cells into cancer stem cells (CSC) leading to over proliferation and drug resistance. This was observed by the nuclear accumulation of YAP/TAZ in several cancer types namely liver, lung, ovary, breast and colon (Yu et al., 2015). Another observation is that YAP/TAZ drives epithelial-to-mesenchymal transition (EMT) leading to an abnormal cell migration, where anoikis is inactive leading to self-renewal of migrating cancer cells, and metastasis (Zhao et al., 2012). This tumorigenic activity of YAP/TAZ is due to its interaction with TEAD, and target genes are upregulated in cancer cells such as glioblastoma, breast cancer and osteosarcoma (Lamar et al., 2012). However, no study has shown a genetic regulation of the Hippo pathway or YAP/TAZ in cancer. In other words, the mutations in the Hippo pathway kinases are very rare in human and no mutations in YAP or TAZ has been observed.
Since the activity of YAP/TAZ is also regulated by other pathways, it is suggested that a deregulation in such pathways can affect the activity of YAP/TAZ to promote cancer (Panciera et al., 2017a). Mutations in GPCR signaling are found in more than 80% of uveal melanoma, with a very high activity of YAP. This also suggests that YAP can be regulated by a mechanical pathway through GPCR signaling and far from genetic alteration. One peculiar example is the activity of YAP as a tumor suppressor rather than an oncogene in hematological cancer such as leukemia, lymphoma and others, due to the frequent deletion of YAP locus. In this case, the inhibition of MST increases apoptosis and inhibits growth (Cottini et al., 2014).
All the mechanisms of regulation of YAP/TAZ in CSC are not well understood and need further study. What is sure is that YAP/TAZ are worthy therapeutic targets in cancer. As mentioned before, a very big problem in chemotherapy today is the resistance that EMT
Statins are also a novel way to treat breast cancer; initially they are drugs that reduce cholesterol in hyper-cholesterol patients. In cancer cells, they inhibit Rho-GTPases activity, which leads to YAP/TAZ inhibition and decrease in the capacity of self-renewal and tumor initiation (Sorrentino et al., 2014).
