Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/b-catenin signaling, we identified a series of acyl hydrazones that act downstream of the b-catenin destruction complex to inhibit both Wnt-induced and cancerassociated constitutive Wnt signaling via destabilization of b-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans. Cancer Res; 71(24); 7628-39. Ó2011 AACR.

Constitutive Wnt signalling is characterized by excessive levels of β-catenin protein and is a frequent occurrence in cancer. APC and Axin are key components of the β-catenin destruction complex that acts to promote β-catenin degradation. The levels of Axin are in turn controlled by tankyrases, members of the PARP-family of poly-ADP-ribosylation enzymes. In colorectal cancer cells, which typically harbor APC mutations, inhibition of tankyrase activity promotes Axin stabilization and attenuates Wnt signalling. Here, we examined the effect of inhibiting tankyrases in breast cancer cells with normal APC. We show that application of the small molecule tankyrase inhibitor, XAV939 or siRNA-mediated abrogation of tankyrase expression increases Axin1 and Axin2 protein levels and attenuates Wnt-induced transcriptional responses in several breast cancer lines. In MDA-MB-231 cells, inhibiton of tankyrase activity also attenuate Wnt3a induced cell migration. Moreover, in both MDA-MB-231 and colorectal cancer cells, XAV939 inhibits cell growth under conditions of serum-deprivation. However, the presence of serum prevents this growth inhibitory effect, although inhibition of Wnt-induced transcriptional and migratory responses was maintained. These results indicate that stabilization of Axin by inhibition of tankyrases alone, may not be an effective means to block tumor cell growth and that combinatorial therapeutic approaches should be considered.

The Hippo pathway is a critical regulator of tissue size, and aberrations in pathway regulation lead to cancer. MST1/2 and LATS1/2 kinases comprise the core of the pathway that, in association with adaptor proteins SAV and MOB, functions in a sequential manner to phosphorylate and inhibit the transcription factors YAP and TAZ. Here we identify mammalian MARK family members as activators of YAP/TAZ. We show that depletion of MARK4 in MDA-MB-231 breast cancer cells results in the loss of nuclear YAP/TAZ and decreases the expression of YAP/TAZ targets. We demonstrate that MARK4 can bind to MST and SAV, leading to their phosphorylation, and that MARK4 expression attenuates the formation of a complex between MST/SAV and LATS, which depends on the kinase activity of MARK4. Abrogation of MARK4 expression using siRNAs and CRISPR/Cas9 gene editing attenuates the proliferation and migration of MDA-MB-231 cells. Our results show that MARK4 acts as a negative regulator of the Hippo kinase cassette to promote YAP/TAZ activity and that loss of MARK4 restrains the tumorigenic properties of breast cancer cells.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.

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The Hippo pathway regulates tissue growth and organ size, and inactivation contributes to cancer. Signals flow through Mst/Lats kinases, which phosphorylate and promote cytoplasmic localization of the transcriptional regulators Yap and Taz to inhibit transcription. Here, we identify the multidomain-containing guanine nucleotide exchange factor (GEF) Arhgef7, or bPix, as a positive Hippo pathway regulator. We show that bPix, which localizes to the cytoplasm, binds both Lats and Yap/Taz and thereby promotes Lats-mediated phosphorylation of Yap/Taz in a GEFindependent manner. bPix is required downstream of both cell density sensing and actin cytoskeletal rearrangements, and we demonstrate that loss of bPix expression in normal mammary epithelial cells strongly reduces Yap/Taz phosphorylation, promotes nuclear localization and increases target gene expression. Conversely, increased expression of bPIX in breast cancer cell lines re-couples the Hippo kinase cassette to Yap/Taz, promoting localization of Yap/Taz to the cytoplasm and inhibiting cell migration and proliferation. These studies thus define bPix as a key component that links the Hippo kinase cassette to Yap/Taz in response to multiple upstream Hippo pathway activators.

A unique nucleus, the cerebrospinal fluid (CSF)-contacting nucleus, has recently been recognized in the brain parenchyma. The outstanding feature of this nucleus is that the neural somas are located in the parenchyma, but their processes stretch into the CSF, implying that it may be a key structure bridging the nervous and body fluids-regulating systems and may play a pivotal role in modulating physiological activities. However, the true biological significance of this nucleus needs to be uncovered. The morphology of a nucleus is one of the most important parameters for neuroscience studies. For this reason, a common experimental animal, Sprague-Dawley (SD) rats, was chosen. The position, adjacent structures, neuronal distribution, size, three-dimensional reconstruction, and core coordinates of the CSF-contacting nucleus in SD rats of different weights (90–400 g) were illustrated for the first time. Furthermore, the formulas for calculating the core coordinates of the CSF-contacting nucleus in rats of different weights were revealed. Finally, the possible biological functions uncovered by past research are reviewed in this paper. This study provides an indispensable methodology and a significant reference for researchers interested in this unique nucleus.