Downstream Effector Of Hippo Pathway Research Articles

Engineered bone marrow mesenchymal stem cell-derived exosomes loaded with miR302 through the cardiomyocyte specific peptide can reduce myocardial ischemia and reperfusion (I/R) injury

BackgroundMicroRNA (miRNA)-based therapies have shown great potential in myocardial repair following myocardial infarction (MI). MicroRNA-302 (miR302) has been reported to exert a protective effect on MI. However, miRNAs are easily degraded and ineffective in penetrating cells, which limit their clinical applications. Exosomes, which are small bioactive molecules, have been considered as an ideal vehicle for miRNAs delivery due to their cell penetration, low immunogenicity and excellent stability potential. Herein, we explored cardiomyocyte-targeting exosomes as vehicles for delivery of miR302 into cardiomyocyte to potentially treat MI.MethodsTo generate an efficient exosomal delivery system that can target cardiomyocytes, we engineered exosomes with cardiomyocyte specific peptide (CMP, WLSEAGPVVTVRALRGTGSW). Afterwards, the engineered exosomes were characterized and identified using transmission electron microscope (TEM) and Nanoparticle Tracking Analysis (NTA). Later on, the miR302 mimics were loaded into the engineered exosomes via electroporation technique. Subsequently, the effect of the engineered exosomes on myocardial ischemia and reperfusion (I/R) injury was evaluated in vitro and in vivo, including MTT, ELISA, real-time quantitative polymerase chain reaction (PCR), western blot, TUNNEL staining, echocardiogram and hematoxylin and eosin (HE) staining.ResultsResults of in vitro experimentation showed that DSPE-PEG-CMP-EXO could be more efficiently internalized by H9C2 cells than unmodified exosomes (blank‐exosomes). Importantly, compared with the DSPE-PEG-CMP-EXO group, DSPE-PEG-CMP-miR302-EXO significantly upregulated the expression of miR302, while exosomes loaded with miR302 could enhance proliferation of H9C2 cells. Western blot results showed that the DSPE-PEG-CMP-miR302-EXO significantly increased the protein level of Ki67 and Yap, which suggests that DSPE-PEG-CMP-miR302-EXO enhanced the activity of Yap, the principal downstream effector of Hippo pathway. In vivo, DSPE-PEG-CMP-miR302-EXO improved cardiac function, attenuated myocardial apoptosis and inflammatory response, as well as reduced infarct size significantly.ConclusionIn conclusion, our findings suggest that CMP-engineered exosomes loaded with miR302 was internalized by H9C2 cells, an in vitro model for cardiomyocytes coupled with potential enhancement of the therapeutic effects on myocardial I/R injury.

TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

The TGF-β and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-β and Hippo pathways, since TGF-β modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-β pathway and TAZ expression and observe that TGF-β induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-β/SMAD-signaling, one of the pathways altered in liver cancer.

FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1.

We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities. Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms. The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK. We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.

Context-dependent transcriptional regulations of YAP/TAZ in cancer.

As the downstream effectors of Hippo pathway, YAP/TAZ are identified to participate in organ growth, regeneration and tumorigenesis. However, owing to lack of a DNA-binding domain, YAP/TAZ usually act as coactivators and cooperate with other transcription factors or partners to mediate their transcriptional outputs. In this article, we first present an overview of the core components and the upstream regulators of Hippo-YAP/TAZ signaling in mammals, and then systematically summarize the identified transcription factors or partners that are responsible for the downstream transcriptional output of YAP/TAZ in various cancers.

Dimethyl Fumarate Combined With Vemurafenib Enhances Anti-Melanoma Efficacy via Inhibiting the Hippo/YAP, NRF2-ARE, and AKT/mTOR/ERK Pathways in A375 Melanoma Cells.

Melanoma is a deadly form of skin cancer with high rates of resistance to traditional chemotherapy and radiotherapy. BRAF inhibitors (BRAFi) can achieve initial efficacy when used to treat melanoma patients, but drug resistance and relapse are common, emphasizing the need for new therapeutic strategies. Herein, we reported that combination of dimethyl fumarate (DMF) and vemurafenib (Vem) inhibited melanoma cell proliferation more significantly and induced more cell death than single agent did both in vitro and in vivo. DMF/Vem treatment induced cell death through inhibiting the expression and transcriptional activity of NRF2 thereby resulting in more reactive oxygen species (ROS) and via inhibiting the expression of YAP, a key downstream effector of Hippo pathway. DMF/Vem treatment also reduced phosphorylation of AKT, 4EBP1, P70S6K and ERK in AKT/mTOR/ERK signaling pathways. RNA-seq analysis revealed that DMF/Vem treatment specifically suppressed 4561 genes which belong to dozens of cell signaling pathways. These results indicated that DMF/Vem treatment manifested an enhanced antitumor efficacy through inhibiting multiple cell signaling pathways, and thus would be a novel promising therapeutic approach targeted for melanoma.

GPNMB promotes the progression of diffuse large B cell lymphoma via YAP1-mediated activation of the Wnt/β-catenin signaling pathway

Glycoprotein non-metastatic melanoma protein B (GPNMB) has been confirmed to be related to the pathogenesis of tumors. However, the potential impact of GPNMB on the progression of diffuse large B-cell lymphoma (DLBCL) is unclear. In this study, the expression levels of GPNMB and Yes-associated protein (YAP) were analyzed using qRT-PCT and Western blot assay. Cell counting kit-8, EdU, and flow cytometry assays were used to detect the proliferation and apoptosis of DLBCL cells. A nude mice xenograft model was established for in vivo research. Results showed that GPNMB and YAP1 were upregulated in DLBCL cell lines. Knockdown of GPNMB inhibited cell proliferation and promoted apoptosis in DLBCL cells. Additionally, the expression levels of YAP1 and the downstream effector of Hippo pathway (c-myc) were markedly decreased when GPNMB was knocked down. Moreover, knockdown of GPNMB inhibited the nuclear translocation of β-catenin protein, which could be abolished by YAP1 overexpression. Simultaneously, the anti-proliferative and pro-apoptotic effects of GPNMB knockdown could be reversed by YAP1 overexpression or LiCl (the activator of Wnt/β-catenin pathway). Furthermore, the mice xenograft model confirmed that inhibition of GPNMB restrained the tumorigenesis of DLBCL in vivo. In conclusion, GPNMB could partly activate the Wnt/β-catenin signaling pathway by targeting YAP1, so as to participate in tumorigenesis of DLBCL.

TAZ-regulated expression of IL-8 is involved in chemoresistance of hepatocellular carcinoma cells

Chemotherapy resistance is one of the major challenges for the treatment of hepatocellular carcinoma (HCC). In order to investigate the mechanisms involved in chemoresistance of HCC, we established cisplatin (CDDP) and doxorubicin (Dox) resistant HCC cells. The expression of transcriptional coactivator with PDZ-binding motif (TAZ), one of the major downstream effectors of Hippo pathway, was upregulated in chemoresistant HCC cells. Targeted inhibition of TAZ via its siRNAs can restore CDDP and Dox sensitivity of chemoresistant HCC cells. The upregulation of TAZ increased the expression of IL-8 in HCC/CDDP and HCC/Dox cells. Recombinant IL-8 (rIL-8) antagonized the increased chemosensitivity mediated by TAZ knockdown. Mechanistically, TAZ can directly bind with the promoter of IL-8 to activate its transcription in chemoresistant HCC cells. Collectively, our data showed that TAZ-regulated expression of IL-8 was involved in chemoresistance of HCC cells. It indicated that targeted inhibition of TAZ/IL-8 axis might be helpful to improve chemotherapy efficiency for HCC.

Anti-oral Squamous Cell Carcinoma Effects of a Potent TAZ Inhibitor AR-42.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Although great progress has been made in diagnosis and treatment strategies in recent years, the 5-year survival rate of OSCC patients is still disappointingly low. Hence, there is still an unmet medical need for sufferers with OSCC. As a downstream effector of Hippo pathway, TAZ was up-regulated in multiple cancers including OSCC, and considered as an effective therapeutic target. In this study, we constructed a stable transfected cell line HEK293-TAZ to screen TAZ inhibitor using dual-luciferase reporter assay, and found a potential TAZ inhibitor AR-42. The results showed that AR-42 effectively suppressed the viability and proliferation of OSCC cells, and induced cellular apoptosis and cell cycle arrest in G2/M phase. Moreover, AR-42 potently inhibited cell invasion and the capacity of sphere-forming, as well as the expression of EMT and cancer stem cell related proteins in OSCC cells, exhibiting potential efficacy against OSCC metastasis and self-renewal of oral cancer stem cell. Further mechanism studies showed that AR-42 inhibited the total amount of TAZ and its paralog YAP mainly through blockade of TAZ/YAP transcription and promotion of TAZ/YAP protein degradation. Additionally, the inhibitory effect of AR-42 against TAZ, as well as its anti-OSCC activity could be also observed in SCC9 xenograft model. Taken together, AR-42 deserves to be further studied as a TAZ inhibitor, and is worthy to be further assessed as a potential drug candidate for OSCC treatment.

LKB1 and YAP phosphorylation play important roles in Celastrol-induced β-catenin degradation in colorectal cancer.

Wnt/β-catenin and Hippo pathways play essential roles in the tumorigenesis and development of colorectal cancer. We found that Celastrol, isolated from Tripterygium wilfordii plant, exerted a significant inhibitory effect on colorectal cancer cell growth in vitro and in vivo, and further unraveled the molecular mechanisms. Celastrol induced β-catenin degradation through phosphorylation of Yes-associated protein (YAP), a major downstream effector of Hippo pathway, and also Celastrol-induced β-catenin degradation was dependent on liver kinase B1 (LKB1). Celastrol increased the transcriptional activation of LKB1, partially through the heat shock factor 1 (HSF1). Moreover, LKB1 activated AMP-activated protein kinase α (AMPKα) and further phosphorylated YAP, which eventually promoted the degradation of β-catenin. In addition, LKB1 deficiency promoted colorectal cancer cell growth and attenuated the inhibitory effect of Celastrol on colorectal cancer growth both in vitro and in vivo. Taken together, Celastrol inhibited colorectal cancer cell growth by promoting β-catenin degradation via the HSF1–LKB1–AMPKα–YAP pathway. These results suggested that Celastrol may potentially serve as a future drug for colorectal cancer treatment.

Histone deacetylase 8 triggers the migration of triple negative breast cancer cells via regulation of YAP signals

Triple-negative breast cancer (TNBC) shows highly aggressive clinical behaviors and poor prognosis compared to other breast cancer subtypes. Histone deacetylases (HDACs) can regulate the progression of various cancers, but the role of HDAC8 in TNBC remains unexplored. Here, we found that HDAC8 enhanced the in vitro migration abilities of breast cancer cells. Targeted inhibition of HDAC8 via si-HDAC8 and its selective inhibitor PCI34051 could suppress the migration of cells. In TNBC cells, HDAC8 stabilized the expression and increased the nuclear localization of YAP, a major downstream effector of Hippo pathway. While silencing YAP could attenuate HDAC8 triggered migration of TNBC cells. Mechanistically, HDAC8 suppressed the phosphorylation of YAPSer127, which was related to its cytoplasmic sequestration degradation. Our data revealed that HDAC8 could trigger the migration of TNBC cells via regulation of Hippo-YAP signals, suggesting that HDAC8 might be a potential target for TNBC therapy.

Overexpression of αCGRP promotes osteogenesis of periodontal ligament cells by regulation of YAP signaling.

Human periodontal ligament cells (hPDLCs) are considered as an ideal cell type for periodontal tissue engineering as hPDLCs own mesenchymal stem cell-like properties. Additionally, it is suggested that α-calcitonin gene-related peptide (αCGRP) plays a pivotal role in the pathogenesis of periodontitis. However, the specific role of αCGRP on the regulation of alveolar bone regeneration which is essential for treatment of periodontitis remains unclear. In this study, lentiviral αCGRP expression vector was first transfected into hPDLCs. αCGRP expression and the osteogenesis-related gene (ALP, RUNX2, OCN, and BSP) expressions were detected. The results showed that expressions of osteogenic phenotypes were upregulated in αCGRP-transfected hPDLCs combined with an increased expression ofYes-associated protein (YAP), which is the key downstream effectors of Hippo pathway. Our observations suggest that αCGRP-mediated hPDLCs' osteogenesis might relate with the activity of YAP signaling. These observations may reflect intrinsic functions of αCGRP in hPDLCs' osteogenesis and its promising role in the treatment of bone deficiency in periodontal regeneration.

Abstract 5498: Natural product-derived carbon nanodots overcome drug resistance through regulation of Hippo pathway effectors

Abstract The yes-associated protein 1 (YAP) is a nuclear downstream effector of Hippo pathway that plays a critical role in various cancers. Literature shows that, in addition to nucleus, YAP also localizes in cytosol and membrane of cells. We and our collaborators previously reported that elevation of YAP associates with Arf in the genetically-engineered mouse (GEM) model of Pten/Trp53, implicating the possible correlation between Hippo/YAP and ARF in human cancers. We recently found ARF is indeed involved in dysregulation of Hippo pathway in prostate cancer. In details, ARF inhibited nuclear YAP translocation in cancer cells, and ARF knockdown suppressed the stability of YAP protein with upregulated active AKT and dysregulated phosphorylation of YAP through ARF/AKT/mTOR/YAP pathway. Here we further report that carbon nanodots (c-dots) can induce DNA damage through an increase of ROS by upregulation of γH2AX and DNA tails in PC3 and DU145 prostate cancer cells. We found that c-dots derived from natural products such as tea or beet can enter into cancer cell nucleus to interact with ARF for the regulation of the ARF-mediated signaling such as the nuclear translocation of YAP. Mechanistically, c-dots bind ARF to induce cell cycle arrest, apoptosis, and PARP cleavage in prostate cancer cells. Most importantly, addition of c-dots into cultured PC3 cells leads to the reversal of drug resistance to mTOR inhibitor Rapamycin and MET inhibitor Crizotinib by enhancing DNA damage. Moreover, c-dots suppress the tumor size and growth of PC3 cells in xenograft mouse model in vivo. Our data suggest that the nano-scale of c-dots derived from natural food may be efficient in overcoming drug resistance via augmentation of DNA damage linked cell death. Thus we reported a new approach of abolishing drug resistance of aggressive malignancy using nanoparticles, implicating an innovative avenue for precision cancer treatment. Citation Format: Ayan A. Nurkesh, Mannix P. Balanay, Tleubek Yeleusizov, Darkhan Tursynkhan, Qing Yang, Xiaoling Wan, Limara Manarbek, Aisulu Maipas, Bakhyt Matkarimov, Yan Zhang, Haiyan Fan, Li-Xia Miao, Si-Pian Li, Zhenbang Chen, Yingqiu Xie. Natural product-derived carbon nanodots overcome drug resistance through regulation of Hippo pathway effectors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5498.

The role of transcriptional coactivator TAZ in gliomas.

The transcriptional coactivator with PDZ-binding motif (TAZ) is one of the important downstream effectors of Hippo pathway. In this study, the potential implication of TAZ in gliomagenesis was explored. TAZ expression was identified to be upregulated in glioma specimens and positively correlated with tumor grade. Meanwhile, its expression in nucleus was increased more significantly with the ascending order of tumor grade. Knocking down TAZ inhibited glioma cell proliferation, invasion and promoted apoptosis. Conversely, enforced upregulation of TAZ promoted proliferation, invasion of glioma cells, and suppressed apoptosis in vitro. When orthotopic glioblastoma mouse model implanted with TAZ knocked down cells, glioma growth was inhibited and survival period was prolonged. Expression of Ki67, MMP-9, Cyclin D1, Bcl-2 and C-myc was varied in accordance with the level of TAZ in glioma cell. The biomarkers of EMT (epithelial-mesenchymal transition), vimentin and N-cadherin, were downregulated when TAZ was suppressed. Using Co-immunoprecipitation TAZ was identified to bind to TEAD4. Therefore, our findings indicate that TAZ is overexpressed in glioma and translocated more into nucleus in high grade glioma. TAZ is involved in gliomagenesis by promoting glioma growth and may benefit to EMT progression. This result suggests that TAZ serves as a potential target for the treatment of glioma.

TAZ promotes cell growth and inhibits Celastrol-induced cell apoptosis.

Hippo pathway is a highly conservative signalling pathway related to the development of organisms, which has been demonstrated to be strongly linked to the tumorigenesis and tumour progression. As the major downstream effector of Hippo pathway, yes-associated protein (YAP), is a transcriptional activator of target genes that are involved in cell proliferation and survival. As an oncogene, YAP can promote cell growth and inhibit cell apoptosis. Another major downstream effector of Hippo pathway, transcriptional co-activators with PDZ-binding motif (TAZ), is nearly 60% homologous with YAP. In the present study, we assume that TAZ probably has the similar function to YAP. To test this issue, we established an inducible and a stable expression system of TAZ in T-Rex-293 and HEK293 cells respectively. The results of cell growth curves, colony formation assay and tumour xenograft growth showed that overexpression of TAZ could promote cell growth invitro and invivo Meanwhile, we found that up-regulated expression of TAZ could partially restore Celastrol-induced cell apoptosis. Induced overexpression of TAZ could up-regulate its target genes including ankyrin repeat domain-containing protein (ANKRD), cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF), increase the expression of B-cell lymphoma-2 (Bcl-2), decrease the expression of Bcl-2 associated X protein (Bax) and activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which may be the mechanism underlying anti-apoptosis of TAZ. All these findings indicated that TAZ acts as an oncogene that could be a key regulator of cell proliferation and apoptosis.

Abstract 1630: Dysregulation of Hippo pathway in metastatic prostate cancer

Abstract Background: Prostate cancer (PCa) is prevalently diagnosed and more than 80% of late stage PCa patients develop bone metastases. We hypothesized that the selective lodging of the PCa cells in the bone can be contributed by increased RANK-mediated cell signaling network. Cancer cell-derived RANKL orchestrates a complex bone-remodeling program facilitating PCa bone colonization through the activation of specific transcription factors (TFs) promoting PCa-bone microenvironment interaction through increased epithelial to mesenchymal transition (EMT), cell growth, and osteomimicry. Overexpression of RNAKL in indolent LNCaP cells drives cancer bone and soft tissue metastases. Methods and Results: Microarray gene expression was performed to compare RANKL- and Neo-vector- transfected LNCaP cells, LNCaPRANKL and LNCaPneo, respectively. qRT-PCR and western blot were performed to confirm the expression of RANKL-targeted genes in these cells. We found the new extracellular diffusible signal mediated by RANKL, regulating Hippo pathway in the aggressive PCa cells. Recombinant RANKL-treated indolent PCa cells, LNCaP and LAPC4, were shown to gain increased aggressiveness. The RANKL-treated indolent PCa cells exhibited down-regulation of Hippo pathway core kinases (Mst1/2 and Lats1/2) but up-regulation of YAP/TAZ expressions. By deleting downstream effectors of Hippo pathway, YAP/TAZ, interrupted the aggressive PCa cells to migrate, proliferate and invade and reversed their EMT phenotype. Inhibitions of YAP/TAZ in aggressive PCa cells via genetic knockdown approach resulted in E-cadherin increase and Vimentin decrease. With the identification of crosstalk between Hippo and TGF-β, or Wnt pathways, this opened up a new network and targeting opportunity for PCa bone metastasis. The metastatic PCa cells with YAP/TAZ deletions were illustrated for sensitization to other therapeutic agents such as TGF-β inhibitors. These results prompted us to study the mechanism, validate the functional significance, and devise potential targeting strategies of Hippo pathway in PCa bone metastasis. Conclusions: Our results showed that dysregulated Hippo pathway in the aggressive PCa cells. With deletions of YAP/TAZ, PCa metastatic abilities were inhibited. Our findings might explain why PCa cells prefer bone as primary metastatic sites. In addition, targeting YAP/TAZ could be a potential therapy for PCa bone metastasis. Citation Format: Jen-Ming Huang, Gina C-Y Chu, Hyung L. Kim, Haiyen E. Zhau, Leland W.K. Chung. Dysregulation of Hippo pathway in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1630.

Abstract 1112: miR-550a-x fuctions as a tumor suppressor by inhibiting the oncogenic YAP1 in human cancers

Abstract Although Yes-associated protein 1 (YAP1), an essential downstream effector of hippo pathway, is a potent oncogene and amplified in human cancers, the overexpression mechanism is unclear. Herein, this study shows that miR-550a-x directly inhibits the oncogenic YAP1 and functions as a tumor suppressor in various human cancer cells. Ectopic overexpression of miRNA-550a-x suppressed proliferation and survival of various cancer cells via inducing apoptosis. miR-550a-x also had a capacity to suppress cell migration, epithelial-mesenchymal transition and tumor sphere formation. Using bioinformatics and microarray analysis, we found YAP1 as a putative target gene. miR-550a-x decreased the mRNA and protein levels of YAP1 in various cancer cells and significantly repressed YAP1 activity with its downstream target genes including CTGF and CYR61. The expression level of miRNA-550a-x were inversely correlated with the YAP1 expression in normal and colorectal cancer samples. Therefore, our data suggest that miRNA-550X functions as a tumor suppressor by targeting YAP1, which may provide a novel therapeutic option for cancer treatment. Citation Format: Min Ho Choe, Joon Kim, Young-Hoon Han, Jae-Sung Kim. miR-550a-x fuctions as a tumor suppressor by inhibiting the oncogenic YAP1 in human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1112.

Abstract A30: Two faces of YAP: Oncogene in malignant progression but barrier for phenotypic transition in LKB1-deficient lung cancer

Abstract Hippo signaling is actively involved in adult tissue homeostasis and cell fate determination. Previous studies have linked the activation of YAP (the major downstream effector of Hippo pathway) with LKB1 deficiency. Here, we characterize the function of YAP in the malignant progression and phenotypic plasticity of LKB1-deficient lung tumors. Using various lung cancer mouse models, we show that YAP expression significantly accelerates lung adenocarcinoma (ADC) malignant progression in KrasG12D mice whereas YAP deletion dramatically delays the process in Lkb1L/L/KrasG12D mice, suggesting that YAP functions as an essential mediator of malignant progression of Lkb1-deficient lung ADC. Further mechanistic studies identified Survivin as the mediator downstream of YAP in promoting malignant progression of Lkb1-deficient lung ADC. Knockdown of Survivin mimics the inhibitory phenotype of YAP knockdown whereas ectopic expression of Survivin rescues the inhibitory function of YAP knockdown. Our previous work has shown LKB1 inactivation confers lung ADC with strong plasticity to progressively change the cell fate and transit to squamous cell carcinoma (SCC). We find that ectopic YAP expression dramatically inhibits ADC to SCC transdifferentiation whereas knockdown of YAP conversely accelerates the transition process. YAP is initially activated by LKB1 loss in ADC, leading to ZEB2 up-regulation in ADC cells, which binds to DNp63 gene promoter to repress DNp63 transcription. During the transition process, extracellular matrix (ECM) depletion in ADC inactivates YAP, thus relieves ZEB2 mediated default repression on DNp63 transcription in ADC, leading to the initiation of squamous differentiation program. Together, these findings uncover the two faces of YAP in lung tumor malignant progression and phenotypic plasticity. YAP is an essential mediator of malignant progression of Lkb1-deficient lung ADC via regulating Survivin whereas an important barrier for lung cancer transdifferentiation through ZEB2 dependent DNp63 repression. Those works shed light on the fundamental role of YAP in regulating cancer progression and lineage phenotypic transition in LKB1 deficient lung tumors, which might help future development of better therapeutic strategies. Citation Format: Wenjing Zhang, Yijun Gao, Xiangkun Han, Fuming Li, Hongbin Ji. Two faces of YAP: Oncogene in malignant progression but barrier for phenotypic transition in LKB1-deficient lung cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A30.

HDAC9 promotes glioblastoma growth via TAZ-mediated EGFR pathway activation.

Histone deacetylase 9 (HDAC9), a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions. We found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.

Abstract 2253: YAP nuclear accumulation involved in drug-induced cellular senescence in vitro

Abstract Cellular senescence is accompanied by some striking morphological changes, such as becoming large, flat and multinucleated. However, how these changes are established is poorly understood. The Hippo pathway plays an important role in the control of organ size. Previous study reported that YAP, one major downstream effector of Hippo pathway, could be regulated by mechanical signals through F-actin bundles. However, whether YAP participates in the control of cell size through cytoskeleton remains unexplained. In our current study, we found that the senescent cells induced with adriamycin or camptothecin became large and flat, and the stress fibers of the senescent cells seemed to maintain or increase, observed by F-actin staining, though in a way of inappropriate actin clumping. Moreover increased nuclear level and activity of YAP were identified in drug-induced senescent cells. Hence, our data indicated that the increased F-actin bundles and mechanical signals could be acquired in senescent cells induced by adriamycin or camptothecin. In summary, based on our findings here, we proposed that YAP might play some roles during drug-induced cellular senescence in vitro. Citation Format: Kai Ma, Shuren Wang, Qing Xu, Mei Liu, Hongxia Zhu, Ningzhi Xu. YAP nuclear accumulation involved in drug-induced cellular senescence in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2253. doi:10.1158/1538-7445.AM2014-2253

CD44 acts through RhoA to regulate YAP signaling

The Hippo pathway plays an important role in both physical and pathogenesis processes. As crucial downstream effectors of Hippo pathway, YAP is inhibited by Lats1/2 through phosphorylation. However, upstream signals that regulate the Hippo pathway have been still poorly understood. Here, we found that knockdown of CD44 reduced YAP expression and nuclear localization, but nearly had no effect on its upstream effectors, Mst1 and Lats1. Downregulated CD44 expression also significantly decreased the expression of YAP downstream effectors CTGF, Cyr61 and EDN1 at mRNA level. Our next study showed that knockdown of CD44 inhibited RhoA expression, which was consistent with RhoA knockdown mediated YAP downregulation. Furthermore, we demonstrated that over expression of the constitutively active RhoA (RhoA-V14) could block the YAP expression decrease mediated by CD44 knockdown. Moreover, downregulation of CD44 significantly promoted cell apoptosis and inhibited cell proliferation, cell cycle progression and migration, which were consistent with the effects of RNAi-mediated YAP knockdown in both A549 and HepG2 cells. Overall, data are presented showing that CD44 could act through RhoA signaling to regulate YAP expression and this study also provide new insights into the regulatory mechanisms of the Hippo–YAP pathway.