YAP Protein Research Articles

Correlation Between Doppler Ultrasound Blood Flow Parameters and Angiogenesis and Proliferation Activity in Breast Cancer.

BackgroundThe aim of this study was to assess the correlation between Doppler ultrasound blood flow parameters and angiogenesis and proliferation activity in breast cancer.Material/MethodsWe enrolled breast cancer patients (n=55) and benign tumor patients (n=40) from Tengzhou Central People’s Hospital from Mar 2014 to Dec 2016. Doppler ultrasound examination was conducted to determine blood flow parameters, and immunohistochemistry (IHC) experiments were performed to determine the protein expression of angiogenesis genes, cell proliferation genes, and tumor-suppressor genes.ResultsCompared with benign tumors, the maximum velocity (Vmax) and resistance index (RI) were significantly different in I–II stage and III–IV stage breast cancer (P<0.01 and P<0.001, respectively). IHC assay showed that VEDGF165, NRP-1, SphK1, CD31, YAP, CTGF, and Gli2 proteins expressions were significantly higher in breast cancer patients (P<0.01 and P<0.001, respectively). PTEN and MFN2 protein expressions of breast cancer patients were significantly lower (P<0.01 or P<0.001, respectively) compared with those of benign tumor patients. VEDGF165, NRP-1, SphK1, CD31, YAP, CTGF, and Gli2 proteins expressions were positively correlated with Vmax and negatively correlated with RI in breast cancer. PTEN and MFN2 protein expressions were negative correlated with Vmax and positively correlated with RI in breast cancer patients.ConclusionsDecreased RI and increased Vmax are correlated with angiogenesis, proliferation, and tumor suppression in breast cancer.

“Verteporfin exhibits anti-proliferative activity in embryonal and alveolar rhabdomyosarcoma cell lines”

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival.YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus.The aim of this study is to evaluate the action of VP on RMS cell lines.This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment.In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

Metformin targets a YAP1-TEAD4 complex via AMPK\u03b1 to regulate CCNE1/2 in bladder cancer cells

BackgroundMetformin has been reported to function as the anti-tumor inhibiting the growth of different types of cancers, including bladder cancer. But there are few reports on the roles of Yap1, the key molecule of Hippo pathway, in the metformin induced inhibition of bladder cancer (BLCA). We are wondering if the inhibitory effect of metformin on bladder cancer is fulfilled via Yap1 and exploring the related mechanism.MethodsMTS and colony formation assays were used to explore the cellular viabilities and proliferation of BLCA cells challenged by metformin at different concentrations, in vitro. Flow Cytometry (FCM) was used to analyze the cell cycle and the cellular apoptosis of the BLCA cells. Western Blot was performed to detect the expressions of AMPKα, Yap1, CCND1, CCNE1/2 and CDK2/4/6 in the metformin-treated BLCA cell lines. RNAi method was used for the related genetic functional analysis. The relationships among Yap1, TEADs and CCNE1/2 were predicted and evaluated using bioinformatics, dual-luciferase reporter and co-immunoprecipitation (Co-IP) assays. For in vivo experiments, a xenograft model was used to investigate the effects of metformin on the proliferation of BLCA cells. And Immunohistochemistry (IHC) assay was performed to assess the expressions of CCNE1/2 and Yap1 proteins in the tumor tissues from the model.ResultsMetformin could inhibit the proliferation of the BLCA cells via inducing the G1 cell cycle arrest without apoptosis. And metformin upregulated the phosphorylated AMPKα and decreased the expressions of Yap1 and CCND1, CCNE1/2 and CDK4/6. AMPK inhibition by compound C (CC) restored the cell proliferation and the G1 cell cycle arrest induced by metformin, in vivo. Knockdown of YAP1 inhibited the proliferation of BLCA cells and caused the cell cycle arrest at G1 phase by decreasing the expressions of CCNE1/2 and other G1 phase related molecules, which has been restored by the Yap 5SA mutant. Bioinformatics analysis showed that trans-factor TEAD4 was highly expressed and positively associated with the expressions of CCNE1 and CCNE2 in BLCA and only TEAD4 was precipitated by Yap1 in the BLCA cells. Further studies demonstrated that Yap1 positively regulated both CCNE1 and CCNE2 expressions via forming complex with TEAD4. Furthermore, we observed that metformin inhibited the cell proliferation by decreasing the expressions of Yap1 and both CCNE1 and CCNE2 in xenograft model.ConclusionsThe results of our study reveal a new potential regulatory pathway in which metformin inhibits cell proliferation via AMPKα/Yap1/TEAD4/CCNE1/2 axis in BLCA cells, providing new insights into novel molecular therapeutic targets for BLCA.

Clinical significance of atypical protein kinase C (PKC\u03b9 and PKC\u03b6) and its relationship with yes-associated protein in lung adenocarcinoma

BackgroundProtein kinase C iota (PKCι) and protein kinase C zeta (PKCζ) are two atypical protein kinase (aPKC) enzymes that contribute to cell proliferation and cancer development. The Hippo/YAP pathway is commonly disrupted and upregulated in cancers. Herein, the expression patterns and clinical relevance of PKCι and PKCζ are evaluated in relation to YAP, a downstream effector of Hippo, in lung adenocarcinoma (LAC). The protein and mRNA expression levels of PKCι, PKCζ, YAP, and their phosphorylated forms, namely p-PKCι, p-PKCζ and p-YAP, are evaluated in relation to clinicopathological factors, including patient survival.MethodsA total of 200 primary LAC tissue samples were examined by immunohistochemistry for PKCι, p-PKCι, PKCζ, p-PKCζ, YAP, and p-YAP protein expression. Sixty pairs of LAC and non-neoplastic lung tissue samples were assessed for PRKCI, PRKCZ, and YAP mRNA levels. PKCι, p-PKCι, PKCζ, and p-PKCζ protein expression were evaluated by Western blot analysis in the PC9 and PC9/GR LAC cell lines with YAP modulation.ResultsLAC demonstrated cytoplasmic PKCι, p-PKCι, PKCζ, and p-PKCζ immunostaining patterns. Positive aPKC protein expressions were related with poor patient survival. Especially, increased p-PKCι protein expression was significantly correlated with higher pathological stage and shortened overall survival. YAP overexpression contributes phosphorylation of PKCι and PKCζ protein expression in the LAC cell line.ConclusionsPKCι and PKCζ are related to YAP in LAC. PKCι and PKCζ play distinct roles in LAC; specifically, p-PKCι overexpression is suggested to underlie factors that indicate a poor prognosis.

Abstract 123: Osteopontin Regulates Adult Cardiomyocyte Division in a Mouse Model of Pressure Overload Induced Heart Failure

Background: Our previous work showed that pharmacological blockade of Osteopontin (OPN) signaling can prevent and reverse heart failure induced by pressure overload in a transverse aortic construction (TAC) mouse model. Surprisingly, OPN Knockout (KO) mice subjected to 3 month TAC had worse cardiac function and bigger hearts than wild type (WT) TAC mice, despite lack of cardiomyocyte hypertrophy. We hypothesized that OPN KO increased adult cardiomyocyte proliferation in TAC-induced heart failure. Methods: Male C57Bl/6 (n=17) or OPN KO (n=11) mice were subject to TAC for 3 months. The protein levels of the mitosis marker H3P was quantified using immunofluorescence in paraffin-embedded myocardial sections. Myocytes were co-stained with WGA and MLC2 to count the number of myocytes per field. Adult primary cardiomyocytes from WT hearts were isolated and analyzed with co-imunoprecipitation (Co-IP) to study the interation between the regeneration factor YAP1, OPN and OPN-regulated proteins such as LDLR. For validation and mechanistic studies, more Co-IP experiments were performed in proliferative human liver HEPG2 cells. To study the effect of OPN blockade on YAP1 nuclear translocation, HEPG2 cells and human iPSC- derived cardiomyocytes (hiPS-CMs) were treated with an IgG or OPN blocking antibody for 24 hours followed by immunostaining for YAP1 and PITX2. Results: Nuclear H3P normalized to myocyte count was significantly increased in OPN KO relative to WT TAC hearts (Fold Change = 1.4; p=0,04). Co-IP results revealed a novel interaction between OPN, LDLR and YAP1. Stimulation of β2 adrenergic receptor increased the formation of this multi-molecular complex in a time-dependent manner. Blockade of OPN by a monocolonal antibody for 24 hours caused nuclear localization of YAP1 and PITX2 in HEPG2 cells and hiPS-CMs. Conclusion: OPN regulates the mitotic program in adult cardiomyocytes. Furthermore, the interaction of OPN with LDLR and YAP1 to form a new multi-molecular protein complex is regulated by β2-cAMP signaling pathway. Importantly, OPN regulates nuclear translocation of the regeneration factors YAP1 and PITX2, suggesting that OPN signaling may be important for adult cardiomyocyte division in TAC and potentially myocardial infarction and aging.

Role of Hippo/YAP signaling in irradiation-induced glioma cell apoptosis.

BackgroundAlthough Hippo/Yes-associated protein (YAP) signaling plays crucial roles in radiation sensitivity and resistance of multiple kinds of cancers, its role in the radiation sensitivity of glioma cells remains unclear. The present study aimed to reveal Hippo/YAP role in the radiation sensitivity of glioma cells.MethodsGlioma U251 cells were administrated with different doses of irradiation. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were used to assess cell viability and apoptosis. Co-immunoprecipitation (co-IP) assay was used to assess the interactions between proteins.ResultsThe results showed that irradiation exposure significantly inhibited cell viability and induced cell apoptosis in a dose-dependent manner, as well as decreased YAP1 expression via enhancing RCHY1-mediated YAP1 protein degradation. In addition, we observed that downregulation of YAP1 or RCHY1 weakened the role of irradiation exposure in cell viability inhibition and apoptosis promotion.ConclusionCollectively, this study emphasizes the vital role of Hippo/YAP signaling in radiation sensitivity of glioma, that RCHY1-mediated YAP1 protein downregulation is a main mechanism accounting for radiation-induced glioma cell apoptosis. Our study may enrich the theoretical basis of Hippo/YAP signaling as a new target for improving radiation sensitivity in glioma.

A Non-canonical Role of YAP/TEAD Is Required for Activation of Estrogen-Regulated Enhancers in Breast Cancer

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an invivo proximity-dependent labeling (BioID) technique, we identified YAP1 and TEAD4 protein as co-regulators of ERα on enhancers. The binding of YAP1/TEAD4 to ERα-bound enhancers is augmented upon E2 stimulation and is required for the induction of E2/ERα target genes and E2-induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. Our data reveal a non-canonical function of YAP1 and TEAD4 as ERα cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERα+ breast cancer.

Targeted Combination Therapy for Glioblastoma by Co-Delivery of Doxorubicin, YAP-siRNA and Gold Nanorods

The combination of brain targeting drug delivery systems and multi-modal intervention pose a promising therapeutic approach for glioblastoma therapy. In this study, we developed an angiopep-2 peptide modified cationic liposomes loaded with doxorubicin, YAP-siRNA and gold nanorods (D/R@Ang2-Lip+Au) simultaneously, which has been characterized by high encapsulating efficiency for doxorubicin (95.4%) and effective binding of siRNA at N/P ratio of 20:1. The fluorescence imaging and flow cytometry analysis revealed high cellular uptake of D/R@Ang2-Lip+Au. Real-time quantitative polymerase chain reaction and western blot analysis indicated that D/R@Ang2-Lip+Au could effectively inhibit the expression of YAP protein. In vitro and in vivo studies showed that D/R@Ang2-Lip+Au had the ability to target glioma cells, and achieved better anti-proliferative effects compared with non-targeted D/R@Lip+Au. Moreover, in vivo experiment demonstrated that D/R@Ang2-Lip+Au was able to cross the blood-brain barrier, and combination therapy could significantly inhibit tumor growth. Therefore, the multifunctional D/R@Ang2-Lip+Au might provide a novel approach for effectively delivery of DOX, AuNRs and siRNA into the glioblastoma cells simultaneously and exerting synergistic therapeutic effects.

Abstract 2886: The Hippo pathway protein YAP mediates resistance to MEK1/2 inhibition in neuroblastoma

Abstract Background: Relapsed NBs harbor an enrichment in mutations activating RAS-MAPK signaling pathway. The MEK1/2 inhibitor trametinib has shown tumor growth delay but not regressions in NB preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed NBs as well as trametinib resistance in other cancers. We hypothesized that increased YAP transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven NBs. Methods: We screened a panel of NB cell lines for mutations predicted to hyperactive the RAS pathway and for high YAP expression. NLF and SK-N-AS cells were treated with the MEK1/2 inhibitor trametinib (20 nM) over 72hrs, and changes in YAP protein expression and cellular localization were observed via immunoblot. YAP protein expression was modulated in NLF, SK-N-AS, and SK-N-FI cells by siRNA-mediated knockdown, CRISPR-Cas9 genetic knockout, and constitutive overexpression. Effects on survival, proliferation, and signaling were observed with and without trametinib treatment by CellTiter-Glo cell cycle analysis, RT-qPCR, and immunoblot. Synergy studies were performed to assess the combination of trametinib and a YAP/TEAD transcriptional activity inhibitor (Vivace Therapeutics) using Incucyte and CellTiter-Glo. Results: In nuclear extracts of NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K), trametinib caused a near-complete translocation of YAP protein into the nucleus after 72 hrs. Transient knockdown and genetic knockouts of YAP in untreated NLF cells had a modest effect by decreasing proliferation by 20% (p&amp;lt;0.01), while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation by 70% (p&amp;lt;0.001). Overexpression of the constitutively-active form of the YAP protein, YAP-5SA, rescued this effect. Finally, synergy assays between trametinib and a YAP-TEAD transcriptional activity inhibitor produced combination index values&amp;lt; 1, indicating strong synergy. Conclusion: Prolonged trametinib exposure causes a marked nuclear enrichment of active YAP, and co-inhibition of MEK and YAP in NB models effectively reduces proliferation and survival. Genetic depletion of YAP in NB cell lines caused a significant increase in sensitivity to trametinib, which was rescued with overexpression of a constitutively-active YAP-5SA. We have identified a potential drug combination to target a YAP-driven mechanism of trametinib resistance in RAS-driven NBs to support future development of a novel combination therapy for NB patients. Citation Format: Grace E. Coggins, Alvin Farrel, Komal Rathi, Colin M. Hayes, Tracy Tang, Leonard Post, John M. Maris. The Hippo pathway protein YAP mediates resistance to MEK1/2 inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2886.

Somatic Hypermutation of the YAP Oncogene in a Human Cutaneous Melanoma.

Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. IMPLICATIONS: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.

Abstract 3581: LncRNA THAP9-AS1 promotes pancreatic cancer growth via sponging miR-484 and interacting with YAP to enhance YAP activity

Abstract This study was supported by grants from the National Natural Science Foundation of China (No. 81872197, 81672616, 81401989 and 81602016); Guangdong Natural Science Funds for Distinguished Young Scholars (No.2016A030306003); Guangdong Special Support Program (No.2017TQ04R809); Science and Technology Program of Guangzhou (No.201710010100 and 201804010001). Abnormal expression of long non-coding RNAs (lncRNAs) has been observed in various cancer types, providing probability to exploit new diagnostic markers, prognostic factors and therapeutic targets in cancer treatment. Our analysis showed lncRNA THAP9-AS1 was highly overexpressed in pancreatic cancer. Here, we aimed to investigate the roles and mechanisms of THAP9-AS1 in pancreatic cancer. We confirmed the overexpression of lncRNA THAP9-AS1 in pancreatic cancer and validated the no protein-coding potential. THAP9-AS1 promotes pancreatic cancer cells growth in vitro and in vivo. Mechanically, THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression disrupts the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown overcomes the effects of THAP9-AS1 overexpression. THAP9-AS1acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds YAP protein and inhibits the phosphorylation mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in pancreatic cancer tissues. THAP9-AS1 and YAP overexpression predicts a poor outcome of pancreatic cancer patients. Our findings indicate that THAP9-AS1 plays an important role in pancreatic cancer growth via enhancing YAP signaling at two aspects. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for pancreatic cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Nan Li, Guohua Yang, Zhimin He, Wanqing Liu, Haiying Liu, Guopei Zheng. LncRNA THAP9-AS1 promotes pancreatic cancer growth via sponging miR-484 and interacting with YAP to enhance YAP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3581.

Abstract 2735: Targeting HDAC prevents smoking-induced pancreatic cancer metastasis

Abstract Background: Cigarette smoking is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Among patients with metastatic PDAC, 90% have liver and 50% have lung metastasis. In this project we investigated the effect of smoking on pancreatic cancer invasion and metastasis in vitro and in vivo. Methods: Pancreatic cancer cells were treated with different doses of 4-(methylnitro-s-amino)-1-(3-pyridyl)-1-butanone (NNK) and cigarette smoke extract (CSE) and cancer cell survival, markers of epithelial to mesenchymal transition (EMT) as well as invasion were measured by MTT assay, Western, RT-PCR and Matrigel Invasion Assay. In a syngeneic mouse model of pancreatic cancer mice were pre-treated with NNK for 4 weeks and mouse cancer cells were injected in the spleen of mice. Progression of the disease including metastasis to liver and lung was monitored by imaging and tissues were collected and analyzed after 6 weeks. Results: Smoking compounds slightly but significantly increased cancer cell survival. They significantly promoted expression of EMT markers and promoted invasion of the cancer cells. Inhibition of histone deacetylase (HDAC) (class I and II), and especially HDAC4, prevented the pro-EMT and invasion effect of smoking compounds. NNK increased the protein level of HDAC4 and the level of Yes-associated protein 1 (Yap) translocated to the nucleus. Inhibition of HDAC4 reversed this effect. Analysis of Yap binding proteins showed presence of HDAC4 in the Yap protein complex. HDAC inhibition decreased the level of Yap-HDAC4 complex formation. In vivo, NNK promoted metastasis of pancreatic cancer cells to mice lung and liver; whereas, inhibition of HDAC decreased the number of metastases to a level lower than control treated mice. Smoking promoted liver and lung fibrosis and inflammation and measured by the level of collagen staining, stellate cells activation and immune cells staining. HDAC inhibition reversed these effects of smoking. Conclusion: We showed for the first time in an animal model that smoking promotes metastasis of PDAC to the lung. We found that HDAC4 is key mediator of this effect. Inhibiting HDAC4 is a promising strategy for preventing PDAC metastasis. Citation Format: Jiyong Yang, Chintan Chheda, Dina Hauptschein, Latifa Zayou, Josiah Tang, Qiang Wang, Stephen J. Pandol, Mouad Edderkaoui. Targeting HDAC prevents smoking-induced pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2735.

Akt plays indispensable roles during the first cell lineage differentiation of mouse.

The first cell lineage differentiation occurs during the development of mouse 8-cell embryo to blastocyst. Akt is a potent kinase whose role during blastocyst formation has not been elucidated. In the present study, immunofluorescence results showed that the Akt protein was specifically localized to the outer cells of the morula. Akt-specific inhibitor MK2206 significantly inhibited mouse blastocyst formation and resulted in decreased expression of the trophectoderm marker Cdx2 and led to granular distribution of ERα in the cytoplasm. Furthermore, knockdown of ERα by siRNA microinjection can also lead to a decrease in the development rate of mouse blastocysts, accompanied by a decrease in the expression level of Yap protein. We conclude that Akt may be indispensable for the first cell lineage differentiation of mouse.

DOWN-REGULATED YAP INHIBITS PROLIFERATION AND MIGRATION OF ORAL SQUAMOUS CELL CARCINOMA CELLS

Objectives The network including Hippo signaling controls growth, proliferation, differentiation and apoptosis of the cell and tissue which also plays crucial role in organ size control in mammals and drosophila. In this network, tumor suppressor kinases include MST and LATS while YAP and TAZ exist as oncoproteins. Above all YAP is associated with the development of early embryo and the regeneration of the skin wound as well as abnormal growth of cancers in case of over-expression. However, there have been no reports on the effect of down-regulation of YAP in oral cancer cells. And further, research is needed to evaluate the role of YAP in oral squamous cell carcinoma(OSCC) cells. In the current study, we investigated the effects of YAP down-regulation on in vitro proliferation and migration of OSCC cells. Methods We screened 13 OSCC cell lines expression of YAP mRNA and protein were confirmed by PCR and western blot analysis. Among them 2 OSCC cell lines (HSC2, KOSCC11), YAP was expressed high levels comparing with other OSCC cell lines. HSC2 and KOSCC11 cell lines were transfected with sh.RNA compared to sh.RNA Control-transfected cells. Also we performed single cell cloning for cell line's clonal isolation. We checked that down-regulation of YAP. And in vitro cell proliferation and migration assays were used to investigate the effect of YAP down-regulation on cell proliferation and migration. Findings The YAP down-regulated OSCC cells grew significantly slower than the sh.RNA Control transfected cells (p Conclusions These results suggest that down-regulation of YAP induces anti-proliferative and anti-migratory effects in OSCC, and YAP may be a useful target molecule for the treatment of OSCC.

PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma.

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient’s tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM.

SOX9 promotes epithelial-mesenchymal transition via the Hippo-YAP signaling pathway in gastric carcinoma cells.

SRY-box 9 (SOX9) is overexpressed in a number of human tumors, including gastric cancer (GC). However, the function of SOX9 in the development of GC remains unknown. In the present study, SOX9 activated the Hippo-yes-associated protein (YAP) signaling pathway to enhance the epithelial-mesenchymal transition in GC cell lines. The results suggested that SOX9 knockdown inhibited invasion, proliferation and migration of GC cells. Furthermore, SOX9 silencing upregulated the expression of E-cadherin, an epithelial marker, and downregulated the expression of mesenchymal markers, including snail family transcriptional repressor 1, vimentin and N-cadherin. SOX9 overexpression increased the expression of the aforementioned markers. SOX9 significantly affected YAP phosphorylation and total YAP protein levels, suggesting that SOX9 is involved in the Hippo-YAP signaling pathway. The current study revealed that SOX9 may be involved in the pathogenesis of GC, and further elucidation of the pathways involved may support the development of novel therapeutic options for the treatment of GC.

Translation of yes-associated protein (YAP) was antagonized by its circular RNA via suppressing the assembly of the translation initiation machinery

Yap is the key component of Hippo pathway which plays crucial roles in tumorigenesis. Inhibition of Yap activity could promote apoptosis, suppress proliferation, and restrain metastasis of cancer cells. However, how Yap is regulated is not fully understood. Here, we reported Yap being negatively regulated by its circular RNA (circYap) through the suppression of the assembly of Yap translation initiation machinery. Overexpression of circYap in cancer cells significantly decreased Yap protein but did not affect its mRNA levels. As a consequence, it remarkably suppressed proliferation, migration and colony formation of the cells. We found that circYap could bind with Yap mRNA and the translation initiation associated proteins, eIF4G and PABP. The complex containing overexpressed circYap abolished the interaction of PABP on the poly(A) tail with eIF4G on the 5′-cap of the Yap mRNA, which functionally led to the suppression of Yap translation initiation. Individually blocking the binding sites of circYap on Yap mRNA or respectively mutating the binding sites for PABP and eIF4G derepressed Yap translation. Significantly, breast cancer tissue from patients in the study manifested dysregulation of circYap expression. Collectively, our study uncovered a novel molecular mechanism in the regulation of Yap and implicated a new function of circular RNA, supporting the pursuit of circYap as a potential tool for future cancer intervention.

Hippo signaling pathway and colorectal cancer

Hippo signaling pathway plays a significant role in the development of colorectal cancer, and acts as an important regulatory pathway which regulates cell proliferation and cell differentiation. Multiple proteins and genes in Hippo signaling pathway, especially the downstream YAP protein, play important roles in the occurrence, metastasis, drug resistance and recurrence of colorectal cancer. YAP and other related genes can be used as targeted markers for predicting drug resistance to colorectal cancer. Hippo signaling pathway gene network can be used as targeted therapy or combination therapy, thus providing new ideas for the treatment of colorectal cancer. Key words: Colorectal neoplasms; Molecular targeted therapy; Hippo signaling pathway; YAP protein

Eupatilin Inhibits Renal Cancer Growth by Downregulating MicroRNA-21 through the Activation of YAP1.

Renal cell carcinoma (RCC) is the second most common human urinary tumor. Eupatilin is the main active ingredient of the traditional Chinese medicine Artemisia asiatica. The effect of Eupatilin on RCC and the underlying mechanism remain unknown. Here, we investigated the anticancer effects and mechanisms of Eupatilin in RCC in vivo and in vitro, laying an experimental foundation for the clinical application of Eupatilin in the treatment of RCC. The results showed that Eupatilin significantly inhibited 786-O cell viability and migration and promoted apoptosis. Eupatilin inhibited the expression of miR-21 in 786-O cells, and overexpression of miR-21 suppressed the effect of Eupatilin on viability, apoptosis, and migration in 786-O cells. Eupatilin inhibited the growth of renal tumors in nude mice by downregulating miR-21. YAP1, which was identified as a target of miR-21, showed significantly lower expression in RCC tissues than in healthy tissues. miR-21 significantly inhibited YAP1 protein expression in 786-O cells and tumor tissues isolated from nude mice, and YAP1 attenuated the effect of miR-21 on the viability, apoptosis, and migration of 786-O cells. In conclusion, Eupatilin inhibited the expression of miR-21, which mediated the proapoptotic and antimigratory effects of Eupatilin by suppressing YAP1 in renal cancer cells. These results suggested that Eupatilin could be a potent agent for the treatment of RCC.

OR02-4 YAP1 Overexpression Associates With Worse Prognosis In Patients With Adrenal Tumors And YAP1 Inhibition By Verteporfin Reduces Tumorigenicity And Reverses Epithelial-mesenchymal Transition In Adrenocortical Tumor Cells

Background: There is no effective adjuvant therapy for patients with advanced adrenocortical tumors (ACT). Abnormal YAP1, a HIPPO pathway effector, is involved in some types of cancers. Recently, we showed association of the overexpression of YAP1 in pediatric ACT (pACT) with worse prognosis, but its association with tumorigenicity and metastasis in ACT or its involvement in adult ACT (aACT) is unknown. Aims:To analyze the association of YAP1 expression in ACT with prognosis and the effect of YAP1 inhibition on tumor growth and epithelial-mesenchymal transition (EMT) in adrenocortical tumor cells.Methods: The YAP1 mRNA expression in pACT and aACT was analyzed using data available in the Gene Expression Omnibus database (GSE76019, ACT=34) and The Cancer Genome Atlas database (ACC, n=76), respectively. In vitro, we treated NCI-H295 adrenocortical tumor cells with Verteporfin (10uM), a FDA-approved drug that inhibits YAP1/TEAD complex. We evaluated mRNA expression of the HIPPO pathway genes (LATS2, STK3, STK4), its targets (CTGF), TEAD4, CTNNB1 and cycle cell progression markers (CCND1, CKD2 and CCNE1) by qPCR. In addition, we analyzed YAP1, phosphoYAP1 (Ser127), Beta-catenin and mesenchymal markers (Vimentin, N-cadherin and the transcription factor, Snail1) protein expression by Western blot (WB). Cell proliferation (MTS), cell invasion (transwell system), and tumorigenesis (soft agar colony formation assay) were assessed after VP treatment. Results:Higher YAP1 mRNA expression was associated with lower disease free survival (DFS) and lower total survival in adults with and with lower DFS in pediatric patients. Verteporfin considerably reduced cell proliferation (-53.2% after 48h and -67.7% after 72h), cell invasion (-85.6%) and the tumorigenicity, shown by reduction of anchorage-independent growth. Verteporfin antitumor effect on ACT cells was shown by reduced mRNA expression of the Beta-catenin encoding gene (CTNNB1; p<0.05), cell cycle progression genes (CCND1, CDK2 and CCNE1; p<0.005) and YAP1 target gene (CTGF) and TEAD4 (p<0.0001), besides increased mRNA expression of core kinase genes of the HIPPO pathway (LATS2, STK3 and STK4; p<0.05). Verteporfin reduced protein levels of YAP1 (-88.5%), phosphoYAP1 (-94.6%) (p<0.0005), Beta-catenin (-50.4%, p<0.001) and the mesenchymal markers Vimentin (-36%, p<0.01), N-cadherin (-66%, p<0.001) and Snail1 (-53%, p<0.05).Conclusions: YAP1 overexpression is a marker in worse prognosis in ACT. YAP1 inhibition by Verteporfin reduced tumor growth through activation of HIPPO pathway, inhibition of cell cycle progression, reduction of Beta-catenin expression, loss of capacity of anchorage-independent growth, reversed EMT by reduction of mesenchymal markers and reduced cell viability in adrenal cells. These results indicate to YAP1 inhibition by Verteporfin as a novel potential adjuvant therapy for patients with ACT.