10413

YAP1

The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. We have recently shown that the human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and that it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. Here, we show that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Because this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition, suggesting that this is a key feature of YAP signaling in mammalian cells.

The purpose of this study is to examine the expression of Yes-associated protein (YAP) in metaplastic carcinoma and compare to those of triple-negative breast carcinoma (TNBC) for investigation of its implication. Tissue microarrays containing 34 cases of metaplastic carcinoma and 175 cases of TNBC were constructed and immunohistochemical staining was used to evaluate expression of the following proteins: YAP and phosphorylated YAP (pYAP). According to immunohistochemical staining results of cytokeratin 5/6, EGFR, claudin 3, claudin 4, claudin 7, E-cadherin, STAT-1, androgen receptor, and GGT-1, metaplastic carcinoma and TNBC were sub-classified into six subtypes: basal-like type, molecular apocrine type, claudin-low type, immune-related type, mixed type, and null type. Comparing the expression of YAP and pYAP in metaplastic carcinoma and TNBC, the expression of nuclear YAP (p = 0.025), cytoplasmic pYAP (p = 0.010), and nuclear pYAP (p = 0.014) in tumor cell was higher in metaplastic carcinoma than TNBC. In metaplastic carcinoma, the nuclear YAP expression in tumor cell was associated with loss of E-cadherin (p = 0.020) and claudin type (p = 0.020), and the stromal YAP expression was associated with claudin 7 positivity (p = 0.003). In conclusion, the YAP expression in metaplastic carcinoma is higher than that in TNBC, representing the association of stemness and epithelial-mesenchymal transition features in metaplastic carcinoma.

PURPOSE: To investigate biological impact of the downregulation of yes-associated protein (YAP) through RNA interference in the process of epithelial-mesenchymal transition in MHCC97H and MHCC97L. METHODS: MHCC97H and MHCC97L cells were transiently transfected by YAP-siRNA. Furthermore, protein expressions and mRNA levels of characteristic markers of epithelial-mesenchymal transition (E-cadherin, N-cadherin) were examined by Western blotting and real-time polymerase chain reaction, and transwell invasion assay was used to detect changes of invasiveness of MHCC97H and MHCC97L cells. RESULTS: The transfected group with YAP-siRNA in MHCC97H after 72 h by Western blotting showed obviously higher expression of E-cadherin compared with the control group (P < 0.05), and lower expression of N-cadherin (P < 0.05). In MHCC97L cells, the expression of E-cadherin was also significantly increased (P < 0.05); however, N-cadherin expression did not significantly change (P > 0.05). Moreover, compared with the control group, Transwell invasion assay showed that the number of the transfected groups was significantly decreased in MHCC97H and MHCC97L cell lines (both P < 0.05). The result of real-time polymerase chain reaction indicated that mRNA levels of E-cadherin increased (P < 0.05), but the mRNA levels of N-cadherin did not significantly change (P > 0.05) in these two cell lines, indicating some effects of post-transcriptional regulation mechanism after silencing YAP. CONCLUSIONS: YAP expression in human hepatocellular carcinoma cell lines MHCC97H and MHCC97L is closely related with the characteristic markers of epithelial-mesenchymal transition, N-cadherin and E-cadherin expression.

Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) co-operate with numerous transcription factors to regulate gene transcriptions. YAP1 and TAZ are negatively regulated by the tumor suppressive Hippo pathway. In human cancers, the Hippo pathway is frequently deregulated and YAP1 and TAZ escape the inhibition by the Hippo pathway. The upregulation of YAP1 and TAZ induces epithelial-mesenchymal transition and increases drug resistance in cancer cells. TAZ is implicated in cancer stemness. In consequence cancers with hyperactive YAP1 and TAZ are associated with poor clinical prognosis. Inhibitors of YAP1 and TAZ are reasoned to be beneficial in cancer therapy. On the other hand, since YAP1 and TAZ play important roles in the regulation of various tissue stem cells and in tissue repair, activators of YAP1 and TAZ are useful in the regenerative medicine. We discuss the potential application of inhibitors and activators of YAP1 and TAZ in human diseases and review the progress of drug screenings to search for them.CI - Copyright(c) Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A-mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors.CI - (c)2016 American Association for cancer Research.

BACKGROUND/AIM: Pancreatic cancer is one of the most threatening and poorly understood human malignancies. MEKK3 (MAP3K3) is a serine/threonine kinase activated by different signaling pathways. YAP and TAZ are critical oncogenic effectors in pancreatic cancer. We hypothesized that MEKK3 could sustain pancreatic cancer by inducing YAP/TAZ oncogenic activities. MATERIALS AND METHODS: In Panc1 and AsPC1 pancreatic cancer cell lines MEKK3 was knocked-out (KO) by the CRISPR/Cas9 method. These cells were used to evaluate MEKK3 contribution to the expression of YAP/TAZ and their target genes, cell migration, stemness, and in vivo tumor growth. RESULTS: MEKK3 KO reduced both EMT and cell migration, the size of 3D colonies and the percentage of CD44(+)/CD24(+)/EpCAM(+) CSC, promoter recruitment of YAP/TAZ and the expression of their target genes. It reduced tumor growth and prolonged mice overall survival. CONCLUSION: Silencing of MEKK3 represents a valid approach to revert in vivo the aggressiveness of pancreatic cancer by modulating YAP/TAZ transcriptional activities.CI - Copyright(c) 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

BACKGROUND: Long noncoding RNAs (LncRNAs) have been reported to be abnormally expressed in human ovarian cancer and associated with the proliferation and metastasis of cancer cells. The objective of this study was to investigate the role and the underlying mechanisms of LncRNA MAP3K20 antisense RNA 1 (MLK7-AS1) in ovarian cancer. METHODS: The expression level of MLK7-AS1 was investigated in human ovarian cancer tissues and cell lines. The effects of MLK7-AS1 knockdown on ovarian cancer cell proliferation, migration, invasion and apoptosis were evaluated in vitro using MTT, colony formation assays, wound healing assays, transwell assays and flow cytometry. Furthermore, the in vivo effects were determined using the immunodeficient NSG female mice. Luciferase reporter assays were employed to identify interactions among MLK7-AS1 and its target genes. RESULTS: In the current study, MLK7-AS1 was specifically upregulated in ovarian cancer tissues and cell lines. Knockdown of MLK7-AS1 inhibited the ability of cell migration, invasion, proliferation, colony formation and wound healing, whereas promoted cell apoptosis in vitro. By using online tools and mechanistic analysis, we demonstrated that MLK7-AS1 could directly bind to miR-375 and downregulate its expression. Besides, MLK7-AS1 reversed the inhibitory effect of miR-375 on the growth of ovarian cancer cells, which might be involved in the upregulation of Yes-associated protein 1 (YAP1) expression. Moreover, knockdown MLK7-AS1 expression inhibited primary tumor growth in ovary and metastatic tumors in multiple peritoneal organs including liver and spleen in vivo, which were partly abolished by miR-375 inhibition. Mechanically, we found that MLK7-AS1 modulated the epithelial-mesenchymal transition (EMT) process by interacting with miR-375/YAP1 both in vivo and vitro, which promoted the expression of Slug. CONCLUSIONS: Taken together, our study showed for the first time that MLK7-AS1 interacted with miR-375 to promote proliferation, metastasis, and EMT process in ovarian cancer cells through upregulating YAP1.

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart diseases. Normally, heart valve mesenchyme is formed from an endothelial to mesenchymal transition (EMT) of endothelial cells of the endocardial cushions. Yes-associated protein 1 (YAP1) has been reported to regulate EMT in vitro, in addition to its known role as a major regulator of organ size and cell proliferation in vertebrates, leading us to hypothesize that YAP1 is required for heart valve development. We tested this hypothesis by conditional inactivation of YAP1 in endothelial cells and their derivatives. This resulted in markedly hypocellular endocardial cushions due to impaired formation of heart valve mesenchyme by EMT and to reduced endocardial cell proliferation. In endothelial cells, TGFbeta induces nuclear localization of Smad2/3/4 complex, which activates expression of Snail, Twist1, and Slug, key transcription factors required for EMT. YAP1 interacts with this complex, and loss of YAP1 disrupts TGFbeta-induced up-regulation of Snail, Twist1, and Slug. Together, our results identify a role of YAP1 in regulating EMT through modulation of TGFbeta-Smad signaling and through proliferative activity during cardiac cushion development.CI - (c) 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Formation of the coronary vasculature is a complex and precisely coordinated morphogenetic process that begins with the formation of epicardium. The epicardium gives rise to many components of the coronary vasculature, including fibroblasts, smooth muscle cells, and endothelium. Hippo signaling components have been implicated in cardiac development and regeneration. However, a role of Hippo signaling in the epicardium has not been explored. Employing a combination of genetic and pharmacological approaches, we demonstrate that inhibition of Hippo signaling mediators Yap and Taz leads to impaired epicardial epithelial-to-mesenchymal transition (EMT) and a reduction in epicardial cell proliferation and differentiation into coronary endothelial cells. We provide evidence that Yap and Taz control epicardial cell behavior, in part by regulating Tbx18 and Wt1 expression. Our findings show a role for Hippo signaling in epicardial cell proliferation, EMT, and cell fate specification during cardiac organogenesis.CI - Copyright (c) 2016 The Authors. Published by Elsevier Inc. All rights reserved.

YAP is a transcriptional co-regulator that plays important roles in various patho-physiological processes, including the survival and death of cells. However, the effect of YAP on apoptosis and EMT, simultaneously mediated by TGF-beta1, is not known. In this study, we demonstrate that YAP can modulate cell fate of apoptosis versus EMT by acting as a surviving factor. Overexpression of YAP in mouse mammary epithelial (NMuMG) cells suppressed TGF-beta1-induced apoptosis, which shifted the cellular response predominantly toward EMT. In contrast, knockdown of YAP induced spontaneous apoptosis and enhanced TGF-beta1-induced apoptosis, leading to a sharp decrease in the proportion of surviving cells that underwent EMT. These data suggest that YAP is an essential factor for modulating cellular responses to TGF-beta1. Further investigation showed that YAP could regulate the expression level and activation of EGFR. Knockdown or inhibition of EGFR abolished the suppressive effect of YAP on apoptosis, whereas activation of EGFR by EGF significantly reduced apoptosis caused by the knockdown of YAP. The results indicate that EGFR and its activation are critical for YAP-mediated suppression of TGF-beta1-induced apoptosis. This study provides a new understanding of the regulatory mechanism underlying the determination of cell fate in response to TGF-beta1-mediated simultaneous apoptosis and EMT.