Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP

School of Biology, University of St Andrews, St Andrews, UK.
Oncogene (Impact Factor: 8.46). 06/2011; 31(2):238-50. DOI: 10.1038/onc.2011.224
Source: PubMed


The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Drosophila melanogaster protein Expanded acts in the Hippo signaling pathway to control organ size. Previously, willin/FRMD6 has been proposed as the human orthologue of Expanded. Willin lacks C-terminal sequences that are present in Expanded and, to date, little is known about the functional role of willin in mammalian cells. When willin is expressed in D. melanogaster epithelial tissues, it has the same subcellular localization as Expanded, but cannot rescue growth defects associated with expanded deficiency. However, we show that ectopic willin expression causes an increase in phosphorylation of the core Hippo signaling pathway components MST1/2, LATS1 and YAP, an effect that can be antagonized by ezrin. In MCF10A cells, loss of willin expression displays epithelial-to-mesenchymal transition features and willin overexpression antagonizes YAP activity via the N-terminal FERM domain of willin. Therefore, in mammalian cells willin influences Hippo signaling activity by activating the core Hippo pathway kinase cassette.

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    • "From these cultures, quantitative RT-PCR demonstrated that the Willin transcript was expressed 10-fold more in the fibroblasts than in the Schwann cells (Figure 1C). Since Willin is an upstream component of the Hippo pathway [9], [11] components of this newly emerging signaling cascade were also analyzed to determine whether they were expressed within the sciatic nerve. Interestingly, MST2 and LATS1 were more strongly expressed within the fibroblasts than Schwann cells, whilst YAP was less expressed and MST1 was present in both cell types at the same level as assessed by quantitative RT-PCR (Figure 1C). "
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    ABSTRACT: Willin/FRMD6 was first identified in the rat sciatic nerve, which is composed of neurons, Schwann cells, and fibroblasts. Willin is an upstream component of the Hippo signaling pathway, which results in the inactivation of the transcriptional co-activator YAP through Ser127 phosphorylation. This in turn suppresses the expression of genes involved in cell growth, proliferation and cancer development ensuring the control of organ size, cell contact inhibition and apoptosis. Here we show that in the mammalian sciatic nerve, Willin is predominantly expressed in fibroblasts and that Willin expression activates the Hippo signaling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These results show that Willin modulates sciatic nerve fibroblast activity indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.
    PLoS ONE 04/2013; 8(4):e60028. DOI:10.1371/journal.pone.0060028 · 3.23 Impact Factor
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    • "Interestingly, genes that play a role in regulating the Hippo pathway were also co-regulated with TAZ-AXL-CTGF. The angiomotin family members act as tumor suppressors by inhibiting the oncogenic functions of YAP and TAZ [44], [77], while FRMD6 also acts as an antagonist of YAP by activating Hippo pathway kinases [78]. In this study, we found that AMOLT1 and FRMD6 are co-overexpressed in TAZ-AXL-CTGF positive tumors, suggesting that AMOLT1 and FRMD6 may form a negative regulatory loop with TAZ activation, which requires further investigation in vitro in colon cancer cell line models. "
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    ABSTRACT: The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer.
    PLoS ONE 01/2013; 8(1):e54211. DOI:10.1371/journal.pone.0054211 · 3.23 Impact Factor
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    • "Overexpression of hYAP1 S127A increased the expression of three Hippo signal transduction pathway genes at both 20 h and 40 h that have been reported to inhibit Yap activity. They are Willin/Frmd6 (Angus et al., 2012), angiomotin-like 2 (amotl2) (Zhao et al., 2011) and ajuba (Das Thakur et al., 2010). After 40 h of doxycycline treatment another putative Yap inhibitor and Hippo pathway member, WWc2 [WW and C2 domain containing 2; kibra homologue (Yoshihama et al., 2012)], is additionally upregulated. "
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    ABSTRACT: Satellite cells are the resident adult stem cells of skeletal muscle. Mitotically quiescent in mature muscle, they can be activated to proliferate and generate myoblasts to supply further myonuclei to hypertrophying or regenerating muscle fibres, or self-renew to maintain the resident stem cell pool. Here, we identify the transcriptional co-factor Yap as a novel regulator of satellite cell fate decisions. Yap expression increases during satellite cell activation and Yap remains highly expressed until after the differentiation versus self-renewal decision is made. Constitutive expression of Yap maintains Pax7(+) and MyoD(+) satellite cells and satellite cell-derived myoblasts, promotes proliferation but prevents differentiation. In contrast, Yap knock down reduces the proliferation of satellite cell-derived myoblasts by ≈40%. Consistent with the cellular phenotype, microarrays show that Yap increases expression of genes associated with Yap inhibition, the cell cycle, ribosome biogenesis and that Yap represses several genes associated with angiotensin signalling. We also identify known regulators of satellite cell function such as BMP4, CD34 and Myf6 (Mrf4) as genes whose expression is dependent on Yap activity. Finally we confirm in myoblasts that Yap binds to Tead transcription factors and co-activates MCAT elements which are enriched in the proximal promoters of Yap-responsive genes.
    Journal of Cell Science 10/2012; 125(24). DOI:10.1242/jcs.109546 · 5.43 Impact Factor
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