Hippo Pathway Review

MST2/Hippo Pathway

Mammalian Sterile Twenty (MST) pathways have been identified as a homologue of the ste20 kinase from Saccharomyces cerevisiae (Creasy and Chernoff 1995). The MST2/Hippo pathway has been identified as a master regulator of cell proliferation, cell death and cell differentiation (Yu and Guan 2013).

The Hippo Pathway in drosophila

In Drosophila, the Hippo pathway was first discovered with the identification of Fat and expanded proteins which were shown to regulate cell proliferation (Boedigheimer and Laughon 1993, Mahoney et al. 1991). The Hippo pathway in Drosophila is composed of a number of core proteins which have homologues in mammalian cells. The Hippo pathway was mapped using genetic screens in Drosophila (Pan 2010). The core components of the pathway include the ste20-like protein kinase Hippo (Hpo), have been shown to promote apoptosis when NIH3T3 cells were treated with phosphatase inhibitors and under stress signals such JNK activators (Creasy and Chernoff 1995).

WART Kinase

The Ser/Thr kinase Warts (Wrt) was identified as a downstream effector of Hippo that functioned in the inhibition of cell growth in Drosophila (Pan 2010). Warts was shown to be a direct substrate of hippo leading to the activation of Warts (Pantalacci et al. 2003). The co-transcription factor Yorkie was identified as a downstream effector of Wrt kinase; phosphorylation of Yorkie lead to inactivation of Wrt kinase. This then inhibits the transcription of genes related to cell growth induced by scalloped transcriptional activity (Huang et al. 2005). The Drosophila ras-association domain family (dRASSF) was identified to compete with salavador for binding to Hippo, which then reduced Hippo activity (Polesello et al. 2006).

The canonical Hippo pathway in mammalians

The Hippo pathway is conserved in mammals but also shows certain divergences in aspects of hippo signalling (Pan 2010). This has led to the idea of a canonical Hippo pathway in mammalians that is organised exactly as the Drosophila Hippo pathway. In this pathway a number of the core proteins of the signalling cascade in Drosophila are conserved in mammalians: MST1/2 (hippo orthologues), Sav1, LATS1/2 (wts orthologues), and Mob1 (Mats orthologues) that form the core kinase cascade that inhibits YAP activity by direct phosphorylation of the Ser127 residue (Yu and Guan 2013).

YAP phosphorylation

The phosphorylation of YAP on the Ser127 results in an increase interaction of YAP with 14-3-3 resulting in YAP being sequestered in the cytoplasm (Zhao et al. 2007). The co-transcription factor YAP/TAZ and the transcription factor TEAD 1-4 (Salvador orthologue) regulate the physiological response of the hippo pathway by the transcription of genes (Zhao et al. 2010). YAP can bind to the transcription factor TEAD inducing the transcription of genes involved in cell growth such as connective tissue growth factor (CTGF) (Zhao et al. 2008).

YAP regulates transcription

Furthermore, YAP regulates transcriptional activity of the transcription factor Runt family member 2 (Runx2) that is involved in osteoblast differentiation (Yagi et al. 1999). The direct upstream elements of the hippo pathway are not well described but proteins involved in hippo activation include two kibra homologs (KIBRA and WWC2), two Mer homologs (FRMD6 and FRMD1), one Mer homolog (NF2/Mer) and two DS homologs (Dchs1 and Dchs2) (Pan 2010). Importantly, the RASSF proteins are not considered part of the canonical Hippo pathway since it has changed its function from an oncogene to an activator of MST2/Hippo proteins.

The canonical Hippo Pathway

The canonical hippo pathway plays a role in the regulation of differentiation via the expression of YAP1 in murine intestine or chick neural tubes (Camargo et al. 2007, Cao et al. 2008). Furthermore, the loss of MST1/2 results in the proliferation of differentiated hepatocytes (Lu et al. 2010). The canonical Hippo pathway regulates apoptosis through the activation of WW45, MST1/2, LATS1/2 and Mob1 that can phosphorylate downstream targets such as histone H2B or transcription factors (FOXO1 or FOXO3) resulting in an apoptotic response (Pan 2010).

Hippo pathway & cancer

Interestingly, the canonical Hippo pathway plays a role in organ development as the MST1/2 knock out mice present an increase in liver size development (Zhou et al. 2009). A number of studies have shown that the canonical Hippo pathway plays a role in the development of cancers and a number of the core components of the hippo pathway deregulated in cancers. MST1/2 expression is downregulated by hypermethylation in soft tissue sarcoma (Seidel et al. 2007), LATS1/2 mRNA are downregulated showing hypermethylation of LATS1 promoter region in human breast cancers (Takahashi et al. 2005) and decrease in expression of Mob1 in colorectal cancer (Kosaka et al. 2007). The data suggest that the downregulation of these tumour suppressors promote carcinogenesis.

The non-canonical Hippo pathway RASSF1A/MST2 pathway in mammals

Although the hippo pathway is also conserved in mammalians, a number of the core proteins seem to have a different function that has led to the view that there are several non-canonical Hippo pathways. As mentioned above, the most obvious difference of the Hippo pathway in mammals compared with Drosophila is the effect of RASSF1A on the MST2 pathway. The signalling pathway triggered by RASSF1A interaction with MST2 triggers a pro-apoptotic signal and it is known as the non-canonical Hippo pathway RASSF1A/MST2 pathway.

RASSF1A pathway

In this pathway, RASSF1A interacts with MST2 via its leucine zipper like the motif known as Sav-RASSF-Hpo (SARAH), and this increases the local concentration of MST2 resulting in the dimerization of MST2 (Agathanggelou et al. 2005, Scheel and Hofmann 2003). The increase in local concentration of MST2 results in trans-phosphorylation of the Thr180 (MST2)/Thr183 (MST1) of the kinase domain leading to activation of MST kinase (Glantschnig et al. 2002). RASSF1A was identified as an activator of MST2 mediated apoptosis. Expression of RASSF1A activates MST2 that activates downstream effectors such as LATS1 Ser/Thr kinase.

LATS1 phosphorylation

The phosphorylation of LATS1 results in its activation. In turn, LATS1 phosphorylates the co-transcription factor YAP1. YAP1 translocates to the nucleus and binds to p73, thereby increasing the transcription of the pro-apoptotic gene puma (Matallanas et al. 2007). The RASSF proteins were observed to promote apoptosis by activating the Hippo pathway in mammals but, compared with dRASSF in Drosophila, appears to have the opposite effect of antagonizing Hippo pathway mediated apoptosis. In mammalian cells the genome codes for 10 RASSF genes.

RASSF1 & Cancer

RASSF1- RASSF6 contain a Ras-association (RA) domain and a SARAH domain that mediates the interaction with MST1/2 (Bao et al. 2011). RASSF1A has been identified as tumour suppressor protein which works by activating MST2 mediated apoptosis and down-regulating the expression of RASSF1A in a number of cancers (lung, breast, prostate, colorectal) by the hypermethylation of CpG islands on the RASSF1 gene (Donninger 2007).

RASSF1 overexpression increases MST1 kinase activity

RASSF1A overexpression was shown to increase the kinase activity of MST1 and this was shown to be involved in Fas stimulation leading to death receptor signalling (Oh et al. 2006). RASSF1A mediates MST2 activated apoptosis as described above by increasing the MST2-LATS1 complex. Therefore, RASSF1A is acting as a scaffold for the MST2 pathway (Matallanas et al. 2007).

YAP1/2

The two homologues of Yorkie in Drosophila found in humans are yes-associated protein (YAP) and tafazzin (TAZ) (Salah et al. 2011). YAP is a co-transcription factor that binds and regulates several different transcription factors. YAP1/2 contains WW, a 14-3-3 binding site, a coiled-coil domain and a PDZ domain that facilitate protein-protein interactions (Pefani et al. 2014). The most striking difference between the Hippo pathway in mammals and in Drosophila is the ability of YAP1 to induce apoptosis or cell growth depending on the stimulus (Bao et al. 2011). YAP1 phosphorylation of the S127 in mammals (S168 Yorkie in Drosophila) leads to 14-3-3 binding and sequesters YAP1 in the cytosol, inactivating its transcriptional activity (Basu et al. 2003).

YAP1

YAP1 was demonstrated in activating apoptosis via interaction with the transcription factor p73 resulting in the transcription of pro-apoptotic gene p53, an upregulated modulator of apoptosis (puma) (Matallanas et al. 2007). The transcription factor transcriptional enhancer factor TEF-1 (TEAD) was identified in YAP1 mediated transcription which results in an increase in transcription of TEAD and connective tissue growth factor (CTGF), therefore resulting in cell growth (Zhao et al. 2008). YAP1 biological functions depend on the upstream input and different stimuli result in different functions of YAP1.