Expression Of YAP Target Genes Research Articles

Tankyrase Inhibitor Sensitizes Lung Cancer Cells to Endothelial Growth Factor Receptor (EGFR) Inhibition via Stabilizing Angiomotins and Inhibiting YAP Signaling

YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/β-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy.

RARγ Downregulation Contributes to Colorectal Tumorigenesis and Metastasis by Derepressing the Hippo-Yap Pathway.

The Hippo-Yap pathway conveys oncogenic signals, but its regulation during cancer development is not well understood. Here, we identify the nuclear receptor RARγ as a regulator of the Hippo-Yap pathway in colorectal tumorigenesis and metastasis. RARγ is downregulated in human colorectal cancer tissues, where its expression correlates inversely with tumor size, TNM stage, and distant metastasis. Functional studies established that silencing of RARγ drove colorectal cancer cell growth, invasion, and metastatic properties both in vitro and in vivo Mechanistically, RARγ controlled Hippo-Yap signaling to inhibit colorectal cancer development, acting to promote phosphorylation and binding of Lats1 to its transcriptional coactivator Yap and thereby inactivating Yap target gene expression. In clinical specimens, RARγ expression correlated with overall survival outcomes and expression of critical Hippo-Yap pathway effector molecules in colorectal cancer patients. Collectively, our results defined RARγ as tumor suppressor in colorectal cancer that acts by restricting oncogenic signaling by the Hippo-Yap pathway, with potential implications for new approaches to colorectal cancer therapy. Cancer Res; 76(13); 3813-25. ©2016 AACR.

A newly identified mechanism involved in regulation of human mesenchymal stem cells by fibrous substrate stiffness

Stiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this study, we fabricated aligned ultrafine fibers and treated the fiber with different annealing temperatures to produce fibrous substrates with different stiffness. Human mesenchymal stem cells (hMSCs) were then cultured on these fibrous substrates. Our results showed that annealing treatment did not change the diameter of electrospun fibers but increased their polymer crystallinity and mechanical properties. The mRNA expression of RUNX2 was upregulated while the mRNA expression of scleraxis was downregulated in response to an increase in substrate stiffness, suggesting that increased stiffness favorably drives hMSCs into the osteogenic lineage. With subsequent induction of osteogenic differentiation, osteogenesis of hMSCs on stiffer substrates was increased compared to that of the cells on control substrates. Cells on stiffer substrates increasingly activated AKT and YAP and upregulated transcript expression of YAP target genes compared to those on control substrates, and inhibition of AKT led to decreased expression of YAP and RUNX2. Furthermore, macrophage migration inhibitory factor (MIF) was increasingly produced by the cell on stiffer substrates, and knocking down MIF by siRNA resulted in decreased AKT phosphorylation. Taken together, we hereby demonstrate that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates hMSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway. Statement of SignificanceStiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this manuscript, we report our new findings that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates human mesenchymal stem cell (hMSC) differentiation through the macrophage migration inhibitory factor-mediated AKT/YAP/RUNX2 pathway. The findings are novel and interesting because we have identified a new mechanism rather than those involving cytoskeleton activity, by which substrate stiffness regulates hMSC behavior.

Atheroprotective laminar flow inhibits Hippo pathway effector YAP in endothelial cells

Atherosclerosis is a mechanobiology-related disease that preferentially develops in the aortic arch and arterial branches, which are exposed to disturbed/turbulent blood flow but less in thoracic aorta where the flow pattern is steady laminar flow (LF). Increasing evidence supports that steady LF with high shear stress is protective against atherosclerosis. However, the molecular mechanisms of LF-mediated atheroprotection remain incompletely understood. Hippo/YAP (yes-associated protein) pathway senses and effects mechanical cues and has been reported to be a master regulator of cell proliferation, differentiation, and tissue homeostasis. Here, we show that LF inhibits YAP activity in endothelial cells (ECs). We observed that YAP is highly expressed in mouse EC-enriched tissues (lung and aorta) and in human ECs. Furthermore, we found in apolipoprotein E deficient (ApoE(-/-)) mice and human ECs, LF decreased the level of nuclear YAP protein and YAP target gene expression (connective tissue growth factor and cysteine-rich protein 61) through promoting Hippo kinases LATS1/2-dependent YAP (Serine 127) phosphorylation. Functionally, we revealed that YAP depletion in ECs phenocopying LF responses, reduced the expression of cell cycle gene cyclin A1 (CCNA1) and proinflammatory gene CCL2 (MCP-1). Taken together, we demonstrate that atheroprotective LF inhibits endothelial YAP activation, which may contribute to LF-mediated ECs quiescence and anti-inflammation.

Human pluripotent stem cell culture density modulates YAP signaling.

Human pluripotent stem cell (hPSC) density is an important factor in self-renewal and differentiation fates; however, the mechanisms through which hPSCs sense cell density and process this information in making cell fate decisions remain to be fully understood. One particular pathway that may prove important in density-dependent signaling in hPSCs is the Hippo pathway, which is regulated by cell-cell contact and mechanosensing through the cytoskeleton and has been linked to the maintenance of stem cell pluripotency. To probe regulation of Hippo pathway activity in hPSCs, we assessed whether Hippo pathway transcriptional activator YAP was differentially modulated by cell density. At higher cell densities, YAP phosphorylation and localization to the cytoplasm increased, which led to decreased YAP-mediated transcriptional activity. Furthermore, total YAP protein levels diminished at high cell density due to the phosphorylation-targeted degradation of YAP. Inducible shRNA knockdown of YAP reduced expression of YAP target genes and pluripotency genes. Finally, the density-dependent increase of neuroepithelial cell differentiation was mitigated by shRNA knockdown of YAP. Our results suggest a pivotal role of YAP in cell density-mediated fate decisions in hPSCs.

Wnt/Beta‐Catenin Cooperates With Yap Signaling To Promote Hepatobiliary Repair In A Model Of Chronic Liver Injury

Proliferation of atypical ductules and compensatory hepatocyte transdifferentiation to cholangiocytes is a hallmark of 3,5‐diethoxycarbonyl‐ 1, 4‐dihydro‐collidine (DDC) diet‐induced injury in mice. Because liver‐specific beta‐catenin conditional KO mice exhibit fewer proliferating ductules after DDC, we hypothesized that Wnt signaling plays a role in this process. To test this, WT animals on control and DDC diet were analyzed for Wnt expression. We found upregulation of 3 Wnts – 7a, 7b, and 10a – in the portal triad of DDC‐fed mice, as determined by tissue microdissection. Cell sorting showed expression of these three Wnts exclusively in the EpCAM+ cell compartment. Addition of Wnt7b and 10a to small cholangiocyte cell line induced proliferation in an autocrine manner, consistent with an increase in CK19+/Ki67+ cells after DDC. In contrast, direct co‐culture of Hek293 cells expressing recombinant Wnt7a significantly increased Sox‐9 gene expression in AML12 cells and primary hepatocytes. To elucidate the role of beta‐catenin in transdifferentiation, we analyzed liver‐specific transgenic (TG) ser45‐mutant b‐catenin‐expressing mice after DDC diet. Compared to WT, TG had significantly upregulated expression of EpCAM and CK19, and more Sox‐9+ hepatocytes. Further, Yap, which regulates liver cell fate, is increased in TG hepatocyte nuclei, concomitant with increased expression of Yap target genes. Thus, Wnt/beta‐catenin signaling cooperates with Yap pathway to regulate hepatobiliary repair after DDC injury.

Identification of Serum-Derived Sphingosine-1-Phosphate as a Small Molecule Regulator of YAP

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.