Hippo signalling in intestinal regeneration and cancer

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ScienceDirect Hippo signalling in intestinal regeneration and cancer Alex Gregorieff1,* and Jeffrey L Wrana1,2 The Hippo pathway is a unique signalling module that regulates cell-specific transcriptional responses and responds to a wide range of intrinsic and extrinsic cues. Besides its classical role in restricting tissue size during development, Hippo signalling is now recognized to control numerous processes including cell proliferation, survival, cell fate determination, epithelial-tomesenchymal transitions and cell migration. Because of its highly dynamic nature, the intestinal epithelium has served as an exceptional model to study the complex roles of Hippo signalling. In this review, we shall present an overview of Hippo function in the mammalian intestine and discuss the various mechanisms regulating Hippo signalling and how they contribute to intestinal regeneration and cancer. Addresses 1 Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Canada 2 Department of Molecular Genetics, University of Toronto, Canada Corresponding author: Wrana, Jeffrey L ([email protected]) Present address: Department of Pathology, McGill University and the Cancer Research Program of the Research Institute of McGill University Health Centre, Montreal, Canada.

*

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response [2–6], while the recent discovery of the Hippo pathway has revealed an additional layer of complexity [7]. The core Hippo pathway in mammals includes the Mst and Lats kinase cassette (see Box 1 for details), which inactivates the transcriptional regulators, Yap and Taz that in turn regulate transcription of genes typically associated with proliferation, cell survival and cell fate. Loss of function studies in vivo have shown that Yap and Taz are dispensable for gut homeostasis, but following chemical injury or gamma irradiation, Yap is required to regenerate the Lgr5+ ISC pool and crypt regeneration [8,9,10]. One exception is the work of Imajo et al., who used a novel intestinal RNAi delivery system [11] and suggested Yap/Taz promote ISC proliferation and differentiation into goblet cells without prior injury. However, the Yap/Taz dependent effects observed in their system might reflect induction of tissue injury responses from the surgical interventions required for virus-mediated gene transfer. Collectively, the studies on Hippo signalling in the gut indicate that under normal circumstances Yap/ Taz are tightly controlled by the inhibitory upstream kinases Mst and Lats that is released during regenerative responses.

This review comes from a themed issue on Cell dynamics Edited by Eugenia Piddini and Helen McNeill

http://dx.doi.org/10.1016/j.ceb.2017.04.005 0955-0674/ã 2017 Elsevier Ltd. All rights reserved.

Introduction A primary function of the gut epithelium is nutrient uptake and forming a protective barrier against the external milieu. To maintain barrier function the epithelium is continuously renewed by intestinal stem cells (ISCs), or crypt base columnar cells (CBCs), located in the crypts of Lieberku¨hn (Figure 1). Over the last decade significant progress has been made in our understanding how ISCs function during homeostasis [1], but less is known about gut epithelial regeneration upon damage and how chronic injury is linked to cancer initiation and progression. In models of gut injury, such as whole body irradiation or dextran sodium sulfate (DSS)-induced colitis, surviving cells of the crypts undergo a rapid, transient proliferative boost that replenishes non-functional cells. Increased Wnt, Egfr and Jak/Stat signalling are important in this www.sciencedirect.com

The initial hyperproliferation phase during crypt regeneration is reminiscent of the effects of Apc loss during tumour initiation and genetic studies show Yap and Taz are also required for adenoma formation in Apcmin mice [10,12,13]. Furthermore, Yap activation by Mst1/2 or Sav deletion increases crypt proliferation and tumorigenicity [9,14], with Yap/Taz potentially regulated by additional Lats-related NDR kinases [15]. Studies in human colorectal cancer cell lines similarly show that reducing Yap/Taz levels leads to impaired proliferation, survival and tumourigenicity [14,16] and a number of clinical reports link high Yap/Taz activity to colorectal cancer progression and overall poor prognosis [17].

Hippo transcriptional program In the crypt epithelium loss and gain of function approaches show that Yap promotes expression of numerous genes implicated in cancer and regenerative signalling (i.e. Egfr ligands, Ctgf, Cyr61, Msln, Il33, etc.) (Figure 1b) [8,10]. Studies in intestinal organoids and tumour initiating cells in vivo, suggest that Yap drives Egfr signalling during crypt regeneration and adenoma formation [10]. Indeed impaired crypt formation in Yap mutant organoids is rescued by the exogenous Egfr ligand, Epiregulin [10]. In human colorectal cancer cells, Yap also promotes anti-apoptotic gene expression (e.g. Bcl2l1 and Birc5) through a transcriptional complex Current Opinion in Cell Biology 2017, 48:17–25

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Figure 1

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Hippo function in the crypt epithelium. (a) The crypt epithelium is subdivided into two functional units: the ISC and transit amplifying (TA) compartments. ISCs reside at the base of the crypts and are intermingled between post-mitotic Paneth cells. The TA compartment is composed of mitotic progenitor cells for the various cell lineages of the gut epithelium (i.e. enterocytes, goblet cells, Paneth cells, enteroendocrine cells, etc.) [86]. The Wnt, Notch, Egfr and Bmp signalling pathways represent the major stimuli regulating ISC/progenitor proliferation, as well as determining cell lineage commitment and differentiation during homeostasis. Receptor activation of these signalling cascades in ISCs is largely dependent on the expression of their specific ligands in the stem cell niche, that is Paneth cells and underlying stromal cells (see arrows) [87–89]. (b) During homeostasis the Mst-Lats kinase cassette, as well as other kinases (i.e. NDR1/2 and Pkcz) maintain Yap in the cytoplasm and transcriptionally inactive. Following tissue injury or an oncogenic event Yap translocates to the nucleus and induces a genetic program that suppresses Paneth cell differentiation and maintenance of ISCs. Representative Yap responsive genes identified so far are highlighted below. The precise mechanism(s) leading to activation of Yap remains unclear (see below). Current Opinion in Cell Biology 2017, 48:17–25

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Hippo signalling in intestinal regeneration and cancer Gregorieff and Wrana 19

Box 1 The Hippo pathway in brief. According to the classic model, activation of Mst leads to phosphorylation of Lats and its adaptor Mob [78]. In turn, activated Lats phosphorylates a pair of transcriptional regulators termed Yap and Taz, which results in their cytoplasmic sequestration and/or degradation through association with 14-3-3 proteins and b-Trcp respectively [79–82]. When the Mst/Lats kinase module is turned off, Yap and Taz remain unphosphorylated and accumulate in the nucleus to drive transcription of genes typically associated with proliferation and cell survival. As a result, unrestricted Yap/Taz activation leads to increased organ size during development, and in adult tissues promotes tumorigenesis [83–85].

with b-catenin and Tbx5 [16]. Yap/Taz signalling in the gut epithelium also leads to induction of both negative (Lats2, Amot) and positive (Tead2 and Taz) Hippo regulatory feedback loops that can fine tune Yap/Taz activity (Figure 1b) [10,18].

Integration of Wnt and Hippo signalling The Hippo pathway appears intimately entwined with the Wnt pathway, as Yap and Taz inhibit Wnt target genes, many of which are known ISC markers [8,10]. Early studies showed that Yap/Taz can sequester Dvl or b-catenin (Figures 1b and 2a) [8,19,20]. More recently Azzolin et al. suggested that Yap/Taz bind to the b-catenin destruction complex via Axin1 (Figure 2b) [21] and in the absence of Wnt stimulation promotes degradation of b-catenin. Interestingly, Yap regulation of b-catenin may depend on the activity of the methyltransferase Setd7, which associates with Axin1, Yap and b-catenin to methylate Yap and facilitate b-catenin activation [22]. Surprisingly, Yap methylation also leads to cytoplasmic retention and inactivation of Yap [23]. Further studies are required to decipher how the seemingly contradictory actions of Setd7 on Yap and b-catenin are compatible with its protumorigenic effects. Finally Park et al. proposed an entirely different mechanism whereby Yap induces secreted Wnt antagonists (Figure 2d) [24] with three of these, Dkk1, Wnt5a and Bmp4, being well known to counteract the effects of canonical Wnt signalling in the crypt [25–27]. Despite this, Yap does not appear to induce these genes in the crypt [8,10], suggesting that this pathway may not be operational in the gut. Besides ‘how’ Hippo signalling inhibits Wnt signalling, an equally important question is ‘why’. Lineage tracing using Lgr5GFP-CreERT mice unequivocally showed Yap is important for post-irradiation ISC maintenance [10], which is seemingly at odds with Yap inhibition of Wnt signalling [10], and that sustained nuclear Yap activity depletes ISCs [8]. However, immediately after damage, Yap regulation is dynamic and transiently suspends the Wnt-driven homeostatic program in Lgr5+ ISCs and prevents excessive Paneth cell differentiation (Figure 1b). Since Wnt maintains the self-renewal capacity of ISCs and promotes Paneth cell differentiation www.sciencedirect.com

[28,29], Hippo-Wnt crosstalk may thus buffer Wnt activity. Indeed, in Yap mutant intestinal organoids, lowering Wnt activity normalized Paneth differentiation and partially rescued mutant organoid morphogenesis [10]. However, inhibition of Wnt/b-catenin by Yap is likely not the sole mechanism leading to suppression of Paneth cells. Indeed in Apc / cells with constitutive b-catenin signalling, Yap loss still elevates Paneth cells both ex vivo and in vivo. Crosstalk between Hippo and Notch may provide an alternative explanation [14], since deleting the Yap inhibitors Mst1/2 enhanced Notch signalling and reduced Paneth cell formation [30]. In conclusion, Yap is not strictly a driver of cell proliferation and survival during regeneration and cancer, but also functions as a key cell fate regulator. Several studies have also suggested that Wnt signalling can in turn stimulate the transcriptional activity of Yap/ Taz (Figures 2 and 3). In the Azzolin model, Yap/Taz not only block b-catenin signalling, but their association with Axin1 and/or b-catenin also maintains them in an inactivate state (Figure 2b) [21,31], with Yap/Taz inhibition partly dependent on Pkcz recruitment, which directly phosphorylates and destabilizes b-catenin and Yap [32]. Wnt-dependent disruption of the b-catenin destruction complex is then proposed to activate Yap/Taz. In a second model, Cai et al. suggested that the tumour suppressor Apc interacts with Lats and Sav1 to promote Lats-mediated phosphorylation and inactivation of Yap (Figure 2c) [12]. Moreover, they propose the Hippo scaf folding properties of Apc are disrupted by GSK-3b inhibition but not Wnt stimulation, as proposed by Azzolin et al. Finally, Park et al. suggested an alternative mechanism whereby Wnts trigger a non-canonical FZD/ROR-Ga12/ 13-RhoGTPases-Lats1/2 pathway that activates Yap/Taz (Figure 2d) [24]. By fully integrating Hippo into the Wnt pathway, these models certainly provide a simple explanation for how Yap/Taz might be activated within gut epithelium. In agreement, Wnt-driven tumours typically show high levels of nuclear Yap. However, in the homeostatic gut neither Yap or Taz are required for Wnt signalling [10,21], suggesting the pathway is not a core Wnt pathway component. Furthermore, as many extrinsic cues regulate Hippo, one cannot exclude that Yap activation in tumours is due to other signals. Indeed, using validated Yap antibodies, it is clear that Yap localization is largely unaffected by acute deletion of Apc in the gut epithelium [10,33]. Accordingly, Yap activation during adenoma initiation likely depends on other signals independent of Wnt activity (see below). Clearly further studies are warranted to resolve these questions.

Regulation of Hippo signalling in the gut Our understanding of downstream events triggered by Yap/Taz in intestinal regeneration and cancer initiation is Current Opinion in Cell Biology 2017, 48:17–25

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Figure 2

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Multiple models of Hippo/Wnt crosstalk. (a) Cytoplasmic Yap/Taz counteract Wnt signalling by sequestering Dvl, thereby preventing CK1d/e mediated phosphorylation of Dvl and downstream b-catenin activation as well as nuclear Dvl-mediated transcriptional activation. Yap/Taz have also been shown to associate and inhibit b-catenin nuclear translocation. (b) Yap/Taz block Wnt signalling by recruiting b-Trcp and promoting b-catenin degradation. Pkcz destabilizes Yap and b-catenin by direct phosphorylation. Upon Wnt stimulation, reorganization of the b-catenin destruction complex leads to the release and nuclear translocation of both b-catenin and Yap. Setd7-mediated methylation of Yap may facilitate b-catenin release, as well as Current Opinion in Cell Biology 2017, 48:17–25

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Hippo signalling in intestinal regeneration and cancer Gregorieff and Wrana 21

Figure 3

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Yap/Taz regulation in the gut epithelium. In this review we discussed four mechanisms regulating activation of Yap/Taz in the gut epithelium: Wnt stimulation, cytokine and Toll receptor signalling, Prostaglandin singalling and mechanotransduction (see text for details). Note that Yap may also feedback on these stimuli to amplify or terminate signalling.

expanding, but our understanding of what regulates Hippo, particularly in vivo, remains limited. This question is a challenge to answer, as Hippo is not regulated like a classical ligand-receptor pathways (e.g. Wnt, Tgfb, Notch, etc.). Rather, the opposite is true, Hippo activity depends on a panoply of input signals including mechanotransduction, G protein-coupled receptors (GPCR), metabolites, receptor tyrosine kinases and more [34]. Below we discuss how some of these may regulate Hippo during gut regeneration and tumorigenesis.

Immune signalling and the Hippo pathway Inflammation and tumourigenesis are linked and may provide a mechanism whereby cytokine signalling is coupled to Yap (Figure 3). To support this notion, Taniguchi et al. showed that overexpression of activated gp130, a common coreceptor of the Il-6 family of inflammatory cytokines, stimulated Yap-dependent crypt hyperproliferation [35]. Furthermore, Il-6 triggered Yap activation via Src family kinases independently of Stat3 and a recent follow up study showed that Yap in turn stimulates expression of Il6ST, the gene encoding gp130 in Apc mutant cells [35,36]. Another key element as yet unexplored, is the impact of pathogens and their metabolites on inflammation and Yap-driven regeneneration and tumorigenesis. As a central driver of gut inflammation determining how the microbiome might intersect with Hippo signalling should prove interesting [37,38].

Indeed, in Drosophila fat bodies stimulation of the innate immune receptor, Toll, activates Hippo signalling, resulting in inactivation of Yki, the fly Yap/Taz ortholog [39]. In turn, activation of Yki stimulates Cactus (or IkB) and vulnerability to Gram-positive bacterial infection. If conserved in mammals, this study would clearly open the door to a whole new facet of Hippo regulation.

Prostaglandins meet Hippo The prostaglandins lie at the crossroads between inflammation, regeneration and tumorigenesis (Figure 3). Prostaglandins are synthesized from archidonic acid by Cox1 and Cox2 enzymes [40,41] and in the gut, prostaglandin E2 (PGE2) protects against DSS-induced colitis, but also promotes tumorigenesis [42,43]. Kim et al. recently demonstrated that PGE2 signals through the Gas-coupled receptor, EP4, to stimulate Yap activity that in turn induces Cox2 and EP4 [44]. Yap also synergized with PGE2 signalling to induce colon tumorigenesis in mice. In addition, studies on types IIA and X phospholipase A2 (PLA2), the former being a well-established modifier of tumorigenicity in the gut, showed another pathway regulating Yap [45]. Secreted forms of PLA2 boost PGE2 production by hydrolyzing phospholipids to generate archidonic acid, whereas the cytoplasmic forms induced Yap expression and phosphorylation. This impaired Wntdriven maturation of Paneth cells with defects in ISC maintenance and regeneration. The prostaglandin

(Figure 2 Legend Continued) negatively regulate Yap activity. (c) The tumour suppressor, Apc, promotes Mst/Lats mediated inhibition of Yap. Inactivation of Apc in colorectal cancer prevents Lats activation and consequently promotes Yap nuclear accumulation and activation. (d) Wnt/Fz/ Ror complex formation stimulates Ga12/13-mediated Rho activation and downstream Lats inhibition. In turn, Yap transcriptional activation promotes expression of secreted Wnt antagonists which inhibit canonical Wnt/b-catenin signalling. www.sciencedirect.com

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22 Cell dynamics

pathway is thus an important modulator of Hippo during gut regeneration and tumorigenesis.

Hippo signalling, mechanotransduction and the tumour microenvironment In the last 5 years, numerous studies have documented how cell shape and stiffness affect cytoskeletal networks and modulate Hippo signalling (Figure 3) [46–50]. In gut organoid cultures, modular synthetic hydrogels with high matrix stiffness promote Yap-dependent ISC cell survival [51], indicating that mechanical stimulation of Yap is likely important in colorectal cancer progression. During adenoma formation the earliest morphological lesions are aberrant crypt foci (ACF), which are enlarged, abnormally-shaped crypts [52]. In mice, Apc-mutant ACFs have high nuclear Yap, whereas earlier lesions with relatively normal morphology showed limited nuclear Yap. In Drosophila, differential cell growth within the wing imaginal disc generates mechanical stress that alters Hippo signalling to control tissue expansion [53]. It is thus intriguing to speculate that Yap activation in ACF may be driven by altered shape and stiffness in growing lesions. Recent technological breakthroughs enabling precise mechanical stimulation of tissues, such as mouse colon, should now provide the tools to test this [54,55]. At later stages, cancer-associated fibroblasts (CAFs) can critically modulate the stiffness of the tumour microenvironment extracellular matrix (ECM) and release various growth factors [56–58,59,60]. In particular, collagen deposition promotes the invasive properties of colon cancer cells [61,62] and together with increased tissue stiffness may modulate Hippo signalling to promote progression. Finally, it should be noted that Hippo signalling not only senses, but also shapes the tumour microenvironment both directly and indirectly. For instance Yap signalling in CAFs promotes matrix stiffening, cancer cell invasion and angiogenesis [63]. Moreover Hippo signalling intersects with the Tgfb pathway [64–66] to influence fibrotic responses in myofibroblasts that is likely to also function during cancer progression [67–69]. Yap regulation of a range of secreted factors may thus broadly function in regenerating crypts and adenomas [10]. For example the Yap target, Il33, has attracted attention for its role in stimulating immune cells and tumour-associated myofibroblasts in the gut [70–72]. Altogether these studies highlight the important role for Hippo signalling in modulating the rich interplay between the tumour microenvironment and cancer cells.

Concluding remarks The discovery and characterization of the Hippo pathway over a decade ago has generated enormous insight into the mechanisms underlying regeneration and cancer in numerous tissues types. Although originally viewed as a potent regulator of cell proliferation and survival, various Current Opinion in Cell Biology 2017, 48:17–25

other functions have now been ascribed to Hippo signalling. In particular, the capacity of Yap/Taz to reprogram cell fate in various tissues besides the gut is now well recognized [73,74,75–77]. In the gut epithelium, the main effectors of the Hippo pathway, Yap and Taz, are effectively shut off by the upstream Mst/Lats kinase cassette and other related kinases. Upon injury or following an oncogenic event, Yap/Taz subcellular localization is subject to dynamic regulation that in the gut is important for ISC maintenance and the formation of intestinal adenomas during tumour initiation. In this context, Yap acts to buffer the prodifferentiation effects of excessive Wnt signalling and also induce expression of proregenerative genes. The last few years has seen significant progress in our understanding of the function and regulation of Hippo signalling in the gut epithelium. Despite these advances many important questions are left unanswered. In this review we highlighted several mechanisms potentially regulating Hippo activity in the gut. However, the relative importance of these upstream signals remains unclear. Moreover the role of the Hippo pathway beyond cancer initiation needs to be further defined. In particular, how does Yap/Taz influence colorectal cancer progression through the adenoma-to-carcinoma transition and metastasis? Finally, is Hippo signalling implicated in other diseases affecting the gastro-intestinal tract, such as inflammatory bowel disease, and can manipulation of this pathway provide a therapeutic avenue?

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31. Azzolin L, Zanconato F, Bresolin S, Forcato M, Basso G, Bicciato S, Cordenonsi M, Piccolo S: Role of TAZ as mediator of Wnt signaling. Cell 2012, 151:1443-1456. 32. Llado V, Nakanishi Y, Duran A, Reina-Campos M, Shelton PM,  Linares JF, Yajima T, Campos A, Aza-Blanc P, Leitges M et al.: Repression of intestinal stem cell function and tumorigenesis through direct phosphorylation of beta-catenin and Yap by PKCzeta. Cell Rep. 2015, 10:740-754. This study demonstrates that Pkcz suppresses intestinal stem cell activity, regeneration and tumorigenesis. The authors suggest that Pkcz associates with components of the b-catenin complex and directly phosphorylates and negatively regulates the levels of Yap and b-catenin. 33. Narimatsu M, Labibi B, Wrana JL, Attisano L: Analysis of Hippo and TGFbeta signaling in polarizing epithelial cells and mouse embryos. Differentiation 2016, 91:109-118. 34. Meng Z, Moroishi T, Guan KL: Mechanisms of Hippo pathway regulation. Genes Dev. 2016, 30:1-17. 35. Taniguchi K, Wu LW, Grivennikov SI, de Jong PR, Lian I, Yu FX,  Wang K, Ho SB, Boland BS, Chang JT et al.: A gp130-Src-YAP module links inflammation to epithelial regeneration. Nature 2015, 519:57-62. Tanigushi et al. demonstrate that Il6/gp130 activates Yap in the gut epithelium via Src family kinases and independently of Stat3. Furthermore, activated gp130 promotes crypt proliferation, regeneration and disrupts Notch-dependent differentiation. This paper provides a foundation for understanding the cause of colitis-associated cancer. 36. Taniguchi K, Moroishi T, de Jong PR, Krawczyk M, Grebbin BM, Luo H, Xu RH, Golob-Schwarzl N, Schweiger C, Wang K et al.: YAP-IL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis. Proc. Natl. Acad. Sci. U. S. A. 2017, 114:1643-1648. 37. Lasry A, Zinger A, Ben-Neriah Y: Inflammatory networks underlying colorectal cancer. Nat. Immunol. 2016, 17:230-240. Current Opinion in Cell Biology 2017, 48:17–25

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48. Bertero T, Oldham WM, Cottrill KA, Pisano S, Vanderpool RR, Yu Q, Zhao J, Tai Y, Tang Y, Zhang YY et al.: Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension. J. Clin. Investig. 2016, 126:33133335.

62. Kai F, Laklai H, Weaver VM: Force matters: biomechanical regulation of cell invasion and migration in disease. Trends Cell Biol. 2016, 26:486-497.

49. Nowell CS, Odermatt PD, Azzolin L, Hohnel S, Wagner EF, Fantner GE, Lutolf MP, Barrandon Y, Piccolo S, Radtke F: Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction. Nat. Cell Biol. 2016, 18:168-180.

63. Calvo F, Ege N, Grande-Garcia A, Hooper S, Jenkins RP, Chaudhry SI, Harrington K, Williamson P, Moeendarbary E, Charras G et al.: Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat. Cell Biol. 2013, 15:637-646.

50. Wang KC, Yeh YT, Nguyen P, Limqueco E, Lopez J, Thorossian S, Guan KL, Li YJ, Chien S: Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis. Proc. Natl. Acad. Sci. U. S. A. 2016, 113:11525-11530. 51. Gjorevski N, Sachs N, Manfrin A, Giger S, Bragina ME, Ordonez Moran P, Clevers H, Lutolf MP: Designer matrices for intestinal stem cell and organoid culture. Nature 2016, 539:560-564. In this study the authors used synthetic hydrogel networks to define the extracellular matrix components required for intestinal stem cell growth in organoid cultures. This study paves the way for a better understanding of how physical forces modulate Hippo signaling. 52. Wargovich MJ, Brown VR, Morris J: Aberrant crypt foci: the case for inclusion as a biomarker for colon cancer. Cancers (Basel) 2010, 2:1705-1716. Current Opinion in Cell Biology 2017, 48:17–25

64. Beyer TA, Weiss A, Khomchuk Y, Huang K, Ogunjimi AA, Varelas X, Wrana JL: Switch enhancers interpret TGF-beta and Hippo signaling to control cell fate in human embryonic stem cells. Cell Rep. 2013, 5:1611-1624. 65. Pefani DE, Pankova D, Abraham AG, Grawenda AM, Vlahov N, Scrace S, O’Neill E: TGF-beta targets the Hippo pathway scaffold RASSF1A to facilitate YAP/SMAD2 nuclear translocation. Mol. Cell 2016, 63:156-166. 66. Varelas X, Samavarchi-Tehrani P, Narimatsu M, Weiss A, Cockburn K, Larsen BG, Rossant J, Wrana JL: The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-beta-SMAD pathway. Dev. Cell 2010, 19:831-844. www.sciencedirect.com

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