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Research Summary

We have studied the transcriptional coactivators, YAP1 and TAZ (also called WWTR1), which are regulated by the tumor suppressive Hippo pathway, and RASSF proteins, which are closely related to the Hippo pathway. For the details, please refer to the attached references.
We have also started to develop the model mice for the study of ageing. We plan to use these mice to clarify the moleculer mechanism underlying ageing-assoicated muscle atrophy (sarcopenia).

The Hippo pathway and transcriptional coactivators, YAP1 and TAZ.

When cells reach confluence or when cells are exposed to DNA damage, the Hippo pathway is activated to phosphorylate YAP1 and TAZ and shut off YAP1- and TAZ-dependent gene transcriptions. Recent studies have revealed that YAP1 and TAZ are regulated by not only the Hippo pathway but also the cell junction proteins, the actin cytoskeleton , and the mechanical stretch (not shown).

The Hippo pathway was originally identified as the signallings that control organ size in Drosophila. The pathway is composed of the upstream regulators (cell adhesion molecules and membrane-associated proteins), the core kinase cascade (two serine/threonine kinases, adaptors, and regulators), and the transcriptional coactivator. The pathway was named after one of the core kinases, Hippo. The basal architecture is well-conserved in mammals. In mammalian Hippo pathway, mammalian Ste20-like kinases (MST1 and MST2, homologues of Hippo) phosphorylate and activate large tumor suppressor kinases (LATS1 and LATS2), which in turn phosphorylate YAP1 and TAZ. YAP1 and TAZ, when phosphorylated, are recruited from the nucleus to the cytoplasm and undergo protein degradation. Thus the Hippo pathway negatively regulates YAP1 and TAZ.

The Hippo pathway-dependent and –independent aspects of RASSF proteins.

RASSF6 and MST2 form the complex and inhibit each other under the basal condition and MDM2 suppresses p53 expression (The upper part).
RASSF6 is released from MST2 upon the activation of the Hippo pathway and enhances self-ubiquitination of MDM2 to up-regulate p53 (The lower part).
RASSF6 also induces apoptosis through p53-independent mechanism (not shown).

Human genome has ten genes that encode proteins with Ras-associated (RA) domains and these gene products are named generally RASSF proteins. RASSF1 to RASSF6 has the RA domains in the middle region followed by the SARAH domain and are called C-RASSFs, whereas RASSF7 to RASSF10 has the RA domain in the N-terminus, lack the SARAH domain, and are called N-RASSFs. Drosophila has one C-RASSF, dRASSF. dRASSF suppresses oncogenesis caused by Ras mutation and is regarded as a tumor suppressor. dRASSF interacts with and inhibits Hippo; namely the tumor suppressor dRASSF antagonizes the tumor suppressor Hippo pathway. This paradoxical finding is not yet fully explained. Mammalian C-RASSFs similarly interact with MST1 and MST2. Among C-RASSF proteins, RASSF1A, a variant of RASSF1, and RASSF2 activate MST2, whereas RASSF6 like dRASSF inhibits MST2. In this point of view C-RASSFs are considered to be regulators of the Hippo pathway. However, C-RASSFs function as tumor suppressors through the Hippo pathway-independent mechanism. For instance, in response to DNA damage, RASSF1A, RASSF3, and RASSF6 enhance the self-ubiquitination of MDM2, stabilize p53, and induce G1/S arrest and apoptosis. We have found that RASSF6 and MST2 form a complex and inhibit each other under the basal condition, but that upon the activation of the Hippo pathway, RASSF6 and MST2 are dissociated. Thereafter the Hippo pathway inhibits YAP1 and TAZ, while RASSF6 induces cell cycle arrest and apoptosis independently. This model may account for the paradoxical finding of dRASSF and the Hippo pathway in Drosophila.

The Hippo pathway and RASSF proteins in human cancers.

In human cancers, the dysfunction of the Hippo pathway, the suppression of C-RASSFs, and gene amplifications of YAP1and TAZ are frequently observed. All these changes lead to the hyperactivity of YAP1 and TAZ, provide cancer cells with malignant properties, and are associated with poor prognosis. Therefore the development of drugs to inhibit YAP1 and TAZ directly or via the Hippo pathway and C-RASSFs is important to control cancers. As the suppression of C-RASSFs delay DNA repair after DNA damage and results in the genomic instability, it is also meaningful to find out how to compensate the functions of C-RASSFs.

Versatile roles of YAP1 and TAZ beyond cancer.

YAP1 and TAZ interact with various transcriptional factors and play versatile roles in physiological and pathophysiological conditions.

YAP1 plays important roles in early embryogenesis and the knockout mice are embryonic lethal. YAP1 is a key molecule that regulates neural and intestinal stem cells and is critical for epidermal stem proliferation. YAP1 protects heart against myocardial ischemic injury. In TAZ knockout mice, the organogenesis of thyroid, lung, and kidney is disturbed. TAZ promotes osteogenesis and myogenesis and inhibits adipogenesis in mesenchymal stem cells. These findings prompt us to speculate that YAP1 activators are useful to facilitate tissue repairs and to maintain brain function. TAZ activators are expected to prevent osteoporosis, muscle atrophy, and obesity.

Chemical and biological approaches to dissect the regulation of YAP1 and TAZ and the functions of C-RASSF proteins.

Examples of the cell-based assays to identify chemical compounds that activate and inhibit YAP1 and TAZ directly or through the Hippo pathway.
The candidate compounds are further assayed by conventional reporter assays and various phenotypic assays such as myogenesis, adipogenesis, cancer cell motility etc.

Recent studies have revealed that YAP1 and TAZ are regulated by cell junction proteins, actin cytoskeleton, and mechanosensory cues. YAP1 and TAZ cross-talk with the signallings of Notch and Wnt. The regulation of YAP1 and TAZ do not solely depend on the phosphorylation by the Hippo pathway. C-RASSFs function as tumor suppressors independently of the Hippo pathway. As discussed above, they regulate p53 but they also induce apoptosis in p53-deficient cells. Our understandings of the Hippo pathway, YAP1, TAZ, and C-RASSF proteins are still far from complete.
To gain further insights, we adopt chemical and biological approaches. We generated various cell-based assays to screen for inhibitors and activators of YAP1 and TAZ. We have obtained several candidate compounds. Some of them regulate YAP1 and TAZ through the Hippo pathway, whereas others work independently of the Hippo pathway. The identification of the molecular targets of these compounds may bring new insights into the regulatory mechanism of YAP1 and TAZ. Needless to say, these candidates provide us with the possibilities to obtain the compounds that are useful to treat various diseases. We are selecting the inhibitors of YAP1 and TAZ that suppress malignant properties of cancer cells in vitro and plan to test them using in vivo transplantation models. We have successfully obtained one TAZ activator that facilitates myogenesis and prevents muscle atrophy. We expect some YAP1 activators to facilitate tissue repairs. The study on C-RASSF functions is more challenging and we still need the basic study to characterize each C-RASSF. Even so we have started to build new methods to monitor C-RASSF expression and plan to discover the compounds that enhance it.