Yes-associated Protein 1 Inhibitor Research Articles

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFβ) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFβ signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFβ2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFβ2 signaling. Immunofluorescence staining showed that TGFβ2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFβ2. In collagen lattice formation assays, the TGFβ2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFβ2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFβ2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFβ2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFβ2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFβ2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

HLF promotes ovarian cancer progression and chemoresistance via regulating Hippo signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, the role of HLF in the regulation of ovarian cancer (OC) remains unknown. Herein, we reported that HLF expression was upregulated in OC tissues and ovarian cancer stem cells (CSCs). Functional studies have revealed that HLF regulates OC cell stemness, proliferation, and metastasis. Mechanistically, HLF transcriptionally activated Yes-associated protein 1 (YAP1) expression and subsequently modulated the Hippo signaling pathway. Moreover, we found that miR-520e directly targeted HLF 3′-UTR in OC cells. miR-520e expression was negatively correlated with HLF and YAP1 expression in OC tissues. The combined immunohistochemical (IHC) panels exhibited a better prognostic value for OC patients than any of these components alone. Importantly, the HLF/YAP1 axis determines the response of OC cells to carboplatin treatment and HLF depletion or the YAP1 inhibitor verteporfin abrogated carboplatin resistance. Analysis of patient-derived xenografts (PDXs) further suggested that HLF might predict carboplatin benefits in OC patients. In conclusion, these findings suggest a crucial role of the miR-520e/HLF/YAP1 axis in OC progression and chemoresistance, suggesting potential therapeutic targets for OC.

Dihydroartemisinin increased the abundance of Akkermansia muciniphila by YAP1 depression that sensitizes hepatocellular carcinoma to anti-PD-1 immunotherapy.

The effect of anti-programmed cell death 1 (anti-PD-1) immunotherapy is limited in patients with hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP1) expression increased in liver tumor cells in early HCC, and Akkermansia muciniphila abundance decreased in the colon. The response to anti-PD-1 treatment is associated with A. muciniphila abundance in many tumors. However, the interaction between A. muciniphila abundance and YAP1 expression remains unclear in HCC. Here, anti-PD-1 treatment decreased A. muciniphila abundance in the colon, but increased YAP1 expression in the tumor cells by mice with liver tumors in situ. Mechanistically, hepatocyte-specific Yap1 knockout (Yap1LKO) maintained bile acid homeostasis in the liver, resulting in an increased abundance of A. muciniphila in the colon. Yap1 knockout enhanced anti-PD-1 efficacy. Therefore, YAP1 inhibition is a potential target for increasing A. muciniphila abundance to promote anti-PD-1 efficacy in liver tumors. Dihydroartemisinin (DHA), acting as YAP1 inhibitor, increased A. muciniphila abundance to sensitize anti-PD-1 therapy. A. muciniphila by gavage increased the number and activation of CD8+ T cells in liver tumor niches during DHA treatment or combination with anti-PD-1. Our findings suggested that the combination anti-PD-1 with DHA is an effective strategy for liver tumor treatment.

Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

YAP1 overexpression contributes to the development of enzalutamide resistance by induction of cancer stemness and lipid metabolism in prostate cancer

Metastatic castration-resistant prostate cancer (mCRPC) is a malignant and lethal disease caused by relapse after androgen-deprivation (ADT) therapy. Since enzalutamide is innovated and approved by US FDA as a new treatment option for mCRPC patients, drug resistance for enzalutamide is a critical issue during clinical usage. Although several underlying mechanisms causing enzalutamide resistance were previously identified, most of them revealed that drug resistant cells are still highly addicted to androgen and AR functions. Due to the numerous physical functions of AR in men, innovated AR-independent therapy might alleviate enzalutamide resistance and prevent production of adverse side effects. Here, we have identified that yes-associated protein 1 (YAP1) is overexpressed in enzalutamide-resistant (EnzaR) cells. Furthermore, enzalutamide-induced YAP1 expression is mediated through the function of chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII) at the transcriptional and the post-transcriptional levels. Functional analyses reveal that YAP1 positively regulates numerous genes related to cancer stemness and lipid metabolism and interacts with COUP-TFII to form a transcriptional complex. More importantly, YAP1 inhibitor attenuates the growth and cancer stemness of EnzaR cells in vitro and in vivo. Finally, YAP1, COUP-TFII, and miR-21 are detected in the extracellular vesicles (EVs) isolated from EnzaR cells and sera of patients. In addition, treatment with EnzaR-EVs induces the abilities of cancer stemness, lipid metabolism and enzalutamide resistance in its parental cells. Taken together, these results suggest that YAP1 might be a crucial factor involved in the development of enzalutamide resistance and can be an alternative therapeutic target in prostate cancer.

Inhibition of Yes-Associated Protein-1 (YAP1) Enhances the Response of Invasive Breast Cancer Cells to the Standard Therapy.

PurposeThe deregulation of the Hippo pathway results in translocation ofYes-associated protein-1 (YAP1) to the nucleus to exert an oncogenic effect. This effect has been demonstrated in several malignancies, yet, in breast cancer (BC), it remains controversial. The present study aimed to investigate the significance of YAP1 expression in BC, its relation to cancer stem cells (CSCs), and the effect of its inhibition on tumor cell survival.Patients and MethodsWe evaluated the expression of YAP1 protein and gene using immunohistochemistry (IHC) and RT-qPCR in FFPE tissue from normal and breast cancer cases. We also studied its association with CSC expression (OCT4, NANOG, and SOX2) and with different clinicopathologic characteristics. Two BC cell lines (MCF7 and MDA-MB-231) were exposed to different concentrations of YAP1 inhibitor “verteporfin” and cell viability was subsequently assessed.ResultsYAP1 mRNA was higher in BC compared to the normal breast tissue (p-value=0.040) and was higher in luminal tumors compared to triple-negative breast cancer (TNBC) (p-value= 0.017). Its expression in tumors was significantly associated with the expression of pluripotency markers (OCT4 and NANOG) (p-value= 0.030 and 0.035, respectively) and its inhibition resulted in a significant reduction of CSC expression in both MCF-7 and MDA-MB-231 cells. YAP1 nuclear expression by IHC, which signifies its activation, was more evident in invasive carcinomas compared to normal breast tissue and in-situ foci where the expression was limited to the cytoplasm. The pretreatment of BC cells (MCF7 and MDA-MB-231) with YAP1 inhibitor “verteporfin” resulted in their sensitization to the effect of tamoxifen and doxorubicin, respectively, and significantly decreased tumor cell proliferation and survival.ConclusionOur results imply that YAP1 is highly expressed and activated in BC and its inhibition could represent a possible novel therapeutic strategy that should be further explored and investigated to improve the outcome of breast cancer patients.

RETRACTED: YAP1 Inhibition in HUVECs Is Associated with Released Exosomes and Increased Hepatocarcinoma Invasion and Metastasis

Hepatocellular carcinoma is one of the most common gastrointestinal malignancies. Anti-angiogenesis therapies have recently demonstrated promise in the treatment of malignancies, although early treatment benefits may be accompanied by metastasis over time. Additional and more effective anti-angiogenic treatment modalities are therefore needed. We previously found that Yes-associated protein 1 (YAP1) expression is increased in hepatocellular carcinoma (HCC), particularly around tumor-associated blood vessels, suggesting a role in angiogenesis. The YAP1 inhibitor verteporfin is presently in anti-angiogenic clinical trials for the treatment of various cancers. Depleted YAP1 from vascular endothelial cells effectively reduced proliferation and tube formation, validating its utility as an anti-angiogenesis target. We also showed that YAP1 depletion or inhibition in vascular endothelial cells leads to increased release of exosomes containing the long non-coding RNA (lncRNA) MALAT1 into the tumor microenvironment. Direct exosomal transfer of MALAT1 to hepatic cells leads to increased hepatic cell invasion and migration via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. These observations may explain the occurrence of distant tumor metastasis with YAP1-associated anti-angiogenic therapy over time. It provides insight into new pathways and treatment paradigms that may be targeted to increase the long-term success of anti-angiogenic therapies.

YAP1 mediates gastric adenocarcinoma peritoneal metastases that are attenuated by YAP1 inhibition

ObjectivePeritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are...

Targeting LncDACH1 promotes cardiac repair and regeneration after myocardium infarction.

Neonatal mammalian heart maintains a transient regeneration capacity after birth, whereas this regeneration ability gradually loses in the postnatal heart. Thus, the reactivation of cardiomyocyte proliferation is emerging as a key strategy for inducing heart regeneration in adults. We have reported that a highly conserved long noncoding RNA (lncRNA) LncDACH1 was overexpressed in the failing hearts. Here, we found that LncDACH1 was gradually upregulated in the postnatal hearts. Cardiac-specific overexpression of LncDACH1 (TG) in mice suppressed neonatal heart regeneration and worsened cardiac function after apical resection. Conversely, in vivo cardiac conditional knockout of LncDACH1 (CKO) and adenovirus-mediated silencing of endogenous LncDACH1 reactivated cardiomyocyte-proliferative potential and promoted heart regeneration after myocardial infarction (MI) in juvenile and adult mice. Mechanistically, LncDACH1 was found to directly bind to protein phosphatase 1 catalytic subunit alpha (PP1A), and in turn, limit its dephosphorylation activity. Consistently, PP1A siRNA or pharmacological blockers of PP1A abrogated cardiomyocyte mitosis induced by LncDACH1 silencing. Furthermore, LncDACH1 enhanced yes-associated protein 1 (YAP1) phosphorylation and reduced its nuclear translocation by binding PP1A. Verteporfin, a YAP1 inhibitor decreased LncDACH1 silencing-induced cardiomyocyte proliferation. In addition, targeting a conserved fragment of LncDACH1 caused cell cycle re-entry of human iPSC-derived cardiomyocytes. Collectively, LncDACH1 governs heart regeneration in postnatal and ischemic hearts via regulating PP1A/YAP1 signal, which confers a novel therapeutic strategy for ischemic heart diseases.

YAP1 inhibits the induction of TNF-α-stimulated bone-resorbing mediators by suppressing the NF-κB signaling pathway in MC3T3-E1 cells.

Yes-associated protein 1 (YAP1), the core downstream effector of the Hippo signaling cascade, was involved in the regulation of osteoblast and osteoclast differentiation and in bone metabolism. However, the regulatory effects and mechanisms of YAP1 on bone-remodeling molecules in osteoblasts under inflammation remain unknown. In this study, YAP1 expression level was downregulated after treatment with inflammatory cytokine tumor necrosis factor-α (TNF-α) in MC3T3-E1 cells. The key osteoclastogenic molecules induced by TNF-α, namely, interleukin-6 and receptor activator of nuclear factor-κB (NF-κB) ligand, were suppressed after lentivirus-induced YAP1 overexpression, which dramatically increased the expression level of osteoprotegerin. Conversely, the expression levels of the above factors showed opposite trends in the YAP1 small interfering RNA and YAP1 inhibitor (verteporfin) group. Mechanistically, YAP1 attenuated the TNF-α-induced activation of the NF-κB signaling pathway as revealed by the reduced expression of phosphorylated-p65 and NF-κB reporter activity and the nuclear translocation of p65. Moreover, the expression level of YAP1 suppressed by TNF-α was reversed by berberine in concentration-dependent manner. Taken together, our study suggests that YAP1 plays a critical role in the regulation of bone metabolism and is a potential therapeutic target for treating inflammatory bone resorption.

Abstract 72: YAP1 mediates initial survival of alectinib therapy in ALK-rearranged lung cancer via pro-apoptotic protein regulation

Abstract Anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib (ALC), have dramatic therapeutic effects on ALK-rearranged lung cancer, but cures are usually not achieved. We focused on tumor cells that survive ALK inhibitor administration and hypothesized that targeted therapy for these cells could provide complete remission. To explore survival factors, we established patient-derived cell lines and screened them using proteome analysis. Three ALK-rearranged ALC-sensitive cell lines (KTOR-1, KTOR-2, KTOR-3) were established from 3 patients; the 50% inhibitory concentrations (IC50)s for ALC were 24-65 nM. Comprehensive protein expression profiles of the 3 cells indicated that exposure to ALC significantly enriched proteins related to actin and extracellular matrix (ECM) adhesion. We focused on Yes-associated protein 1 (YAP1), which is activated by ECM adhesion and actin fiber accumulation. Nuclear localization of YAP1 (an activation marker of YAP1) was assessed using immunohistostaining. In KTOR1-3 and H2228 cells from an ALK-rearranged line purchased from ATCC, exposure to ALC in vitro promoted YAP1 accumulation in the nucleus. BALB/nu mice xenograft models of H2228 or KTOR1 were administered ALC (8 mg/kg/day, N=4) or a vehicle (N=4) for 7 days, and tumors were evaluated. In ALC-administered tumors, YAP1 was localized to the nucleus, which was rarely the case in vehicle-administered tumors. The expression of pro-apoptosis factors Mcl-1 and Bcl-xL also increased after exposure to ALC in vitro, but the increment was cancelled by YAP1 inhibition by siRNA or verteporfin (VER), a non-specific YAP1 inhibitor. Exposure to ALC with combinatorial YAP1 inhibition significantly increased Caspase 3/7 activity. To address the treatment effects of YAP1 inhibition, a YAP1-activated H2228 cell line (H2ARY) was established by exposing H2228 cells to 100-300 nM of ALC for 3 months and thorough subsequent cloning. The H2ARY had lower sensitivity to ALC in vitro than parental H2228 (IC50: 1.4 μM vs 315 nM, 96 h) and restored the sensitivity by YAP1 inhibition (208 nM with VER 1 μM, 312 nM with siYAP1). Twenty-four xenograft models (mean volume: 199 mm3) of H2ARY on BALB/nu mice were randomized (Day 0) into 4 treatment groups to receive ALC monotherapy (8 mg/kg daily, N=6), VER monotherapy (12.5 mg/kg twice a week, N=7), combination (N=7), or vehicle (N=5). On day 15, the tumor volume of the vehicle and VER monotherapy groups reached > 800 mm3, with no significant differences among the groups. On day 33, the tumors of the combination group were significantly smaller than those of the ALC monotherapy group (187 vs 761 mm3, P = 0.0125). Exposure to ALC-activated YAP1 may regulate anti-apoptotic activity by controlling the expression of Mcl-1 and Bcl-xL in ALK-rearranged lung cancer cells. This is the first evidence that combinatorial therapy against ALK and YAP1 could enhance ALK-rearranged tumor treatment. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Wataru Aoki, Shunsuke Aburaya, Tomoko Funazo, Koh Furugaki, Yasushi Yoshimura, Hitomi Ajimizu, Yuto Yasuda, Takashi Nomizo, Yuichi Sakamori, Hironori Yoshida, Mitsuyoshi Ueda, Young Hak Kim, Toyohiro Hirai. YAP1 mediates initial survival of alectinib therapy in ALK-rearranged lung cancer via pro-apoptotic protein regulation [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 72.

Verteporfin suppresses the proliferation, epithelial-mesenchymal transition and stemness of head and neck squamous carcinoma cells via inhibiting YAP1

Purpose: Yes-associated protein 1 (YAP1) is overexpressed in head and neck squamous cell carcinoma (HNSCC). However, it is unknown whether verteporfin, a YAP1 inhibitor, can inhibit HNSCC cells as well as the molecular mechanisms involved.Methods: YAP1 expression was investigated by immunohistochemistry in human head and neck carcinoma tissues (n=70). CCK-8 assay, colony formation assay, flow cytometric analysis, wound-healing assay and Transwell migration and invasion assays were used to evaluated the effects of verteporfin on the six HNSCC cell lines (three HPV-positive and three HPV-negative). The transcription and protein expression levels of YAP1 and its associated genes were investigated by real-time PCR and Western blotting, respectively. The effects of verteporfin on HNSCC cells in vivo were assessed by a xenograft model.Results: YAP1 expression was significantly higher in carcinoma tissues than in tumor-adjacent normal tissues (n=10). A CCK-8 assay showed that the inhibitory effects of verteporfin on HNSCC cells were markedly enhanced by light activation. Verteporfin significantly inhibited HNSCC cell proliferation, migration and invasion, induced apoptosis, and arrested the cell cycle at the S/G2 phase. Verteporfin significantly attenuated the expression of genes related to epithelial-mesenchymal transition (YAP1, Snail, CTNNB1 and EGFR) and stemness (Oct4 and YAP1) and increased E-cadherin expression in HNSCC cells. Furthermore, verteporfin significantly inhibited PD-L1 expression in HNSCC cells. However, the expression levels of HPV-16 E6 and E7 did not change with VP treatment. The anticancer effect of verteporfin on HNSCC was confirmed by the inhibition of xenograft growth in vivo.Conclusions: Our results indicate that YAP1 overexpression is involved in HNSCC tumorigenesis and verteporfin is a potential therapeutic drug for HNSCC.

Abstract 3962: Antitumor effects of the antiparasitic agent ivermectin via inhibition of yes-associated protein 1 expression in gastric cancer

Abstract [Background] Yes-associated protein 1 (YAP1) acts as an oncogene through dephosphorylation and nuclear translocation, and nuclear accumulation of YAP1 is associated with poor prognosis in gastric cancer (GC). We previously identified ivermectin, an antiparasitic drug, as a YAP1 inhibitor. Here, we aimed to clarify whether ivermectin had antitumor effects on GC through inhibition of YAP1. [Result] First, we evaluated the antiproliferative effects of ivermectin on human GC cells using in vitro proliferation assays and a xenograft mouse model. YAP1-knockdown assays were performed to assess whether the sensitivity to ivermectin depended on YAP1 expression. Next, we explored the mechanism through which ivermectin regulated YAP1 expression or localization by immunoblotting and reverse transcription-quantitative polymerase chain reaction for YAP1 and the downstream gene CTGF. Finally, the clinical significance of YAP1 expression was examined using three independent GC datasets. We found that MKN1 GC cells were most sensitive to ivermectin, whereas MKN7 cells were most resistant. In MKN1 xenografts, ivermectin suppressed tumor growth, and the sensitivity of MKN1 cells to ivermectin was decreased by YAP1 knockdown. Ivermectin inhibited YAP1 nuclear expression and CTGF expression in MKN1 cells but not MKN7 cells. Moreover, ivermectin decreased YAP1 mRNA expression, thereby inhibiting nuclear accumulation of YAP1 in MKN1 cells. In survival analysis, low YAP1 mRNA expression was associated with a better prognosis in three independent GC datasets. [Conclusion] Ivermectin suppressed the growth of GC in vitro and in vivo by inhibiting YAP1 expression. Furthermore, GC with low YAP1 expression had favorable clinicopathological features and good prognosis. These findings provided insights into the antiproliferative effects of ivermectin as a YAP1 inhibitor and established a theoretical basis for preclinical evaluations of ivermectin for management of GC. Citation Format: Shotaro Kuramitsu, Sho Nanbara, Qingjiang Hu, Yousuke Kuroda, Takaai Masuda, Shuhei Ito, Hidetoshi Eguchi, Yoshihiko Mehara, Koshi Mimori. Antitumor effects of the antiparasitic agent ivermectin via inhibition of yes-associated protein 1 expression in gastric cancer [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 3962.

The Hippo/YAP1 pathway interacts with FGFR1 signaling to maintain stemness in lung cancer

The Hippo pathway plays a critical role in organ size control, tissue homeostasis and tumor genesis through its key transcription regulator Yes-associated protein1 (YAP1), but the mechanism underlying its role in lung cancer is unclear. We hypothesized that YAP1 influences FGFR1 signaling to maintain cancer stem-like cell (CSC) properties in FGFR1-amplified lung cancer. In support of this, our data confirms that expression levels of YAP1 are positively associated with those of FGFR1 in clinical lung carcinoma samples as measured by real-time PCR, western blot, and immunohistochemistry (IHC) staining. Mechanistically, YAP1 up-regulates FGFR1 expression at the level of promoter through the TEAD binding site while bFGF/FGFR1 induces YAP1 expression via large tumor suppressors 1(LATS1). In addition, the absence of YAP1 abolishes self-renewal ability in lung cancer. Furthermore, an orthotropic mouse model highlights the function of YAP1 in the initiation and metastasis of lung cancer. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and FGFR1 expression in lung cancer. Thus, we conclude that YAP1 is a potential therapeutic target for lung cancer. Combined targeting of YAP1 and FGFR1 may provide benefits to patients with FGFR1-amplified lung cancer.

A Novel YAP1 Inhibitor Targets CSC-Enriched Radiation-Resistant Cells and Exerts Strong Antitumor Activity in Esophageal Adenocarcinoma.

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high-expressing esophageal adenocarcinoma cells both in vitro and in vivo Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443-54. ©2017 AACR.

Antitumor effects of the antiparasitic agent ivermectin via inhibition of Yes-associated protein 1 expression in gastric cancer.

Yes-associated protein 1 (YAP1) acts as an oncogene through dephosphorylation and nuclear translocation, and nuclear accumulation of YAP1 is associated with poor prognosis in gastric cancer (GC). We previously identified ivermectin, an antiparasitic drug, as a YAP1 inhibitor. Here, we aimed to clarify whether ivermectin had antitumor effects on GC through inhibition of YAP1. First, we evaluated the antiproliferative effects of ivermectin on human GC cells using in vitro proliferation assays and a xenograft mouse model. YAP1-knockdown assays were performed to assess whether the sensitivity to ivermectin depended on YAP1 expression. Next, we explored the mechanism through which ivermectin regulated YAP1 expression or localization by immunoblotting and reverse transcription-quantitative polymerase chain reaction for YAP1 and the downstream gene CTGF. Finally, the clinical significance of YAP1 expression was examined using three independent GC datasets. We found that MKN1 GC cells were most sensitive to ivermectin, whereas MKN7 cells were most resistant. In MKN1 xenografts, ivermectin suppressed tumor growth, and the sensitivity of MKN1 cells to ivermectin was decreased by YAP1 knockdown. Ivermectin inhibited YAP1 nuclear expression and CTGF expression in MKN1 cells but not MKN7 cells. Moreover, ivermectin decreased YAP1 mRNA expression, thereby inhibiting nuclear accumulation of YAP1 in MKN1 cells. In survival analysis, low YAP1 mRNA expression was associated with a better prognosis in three independent GC datasets. In conclusion, we identified ivermectin as a potential antitumor agent and found a promising novel therapeutic strategy for inhibition of GC progression by blocking YAP1 expression.

Targeting hypoxia-mediated YAP1 nuclear translocation ameliorates pathogenesis of endometriosis without compromising maternal fertility.

Endometriosis is a highly prevalent gynaecological disease that severely reduces women's health and quality of life. Ectopic endometriotic lesions have evolved mechanisms to survive in the hypoxic peritoneal microenvironment by regulating the expression of a significant subset of genes. However, the master regulator controlling these genes remains to be characterized. Herein, by using bioinformatics analysis and experimental verification, we identified yes-associated protein 1 (YAP1) as a master regulator of endometriosis. Nuclear localization and transcriptional activity of YAP1 were up-regulated by hypoxia via down-regulation of LATS1, a kinase that inactivates YAP1. Disruption of hypoxia-induced YAP1 signalling by siRNA knockdown or inhibitor treatment abolished critical biological processes involved in endometriosis development such as steroidogenesis, angiogenesis, inflammation, migration, innervation, and cell proliferation. Treatment with a YAP1 inhibitor caused the regression of endometriotic lesions without affecting maternal fertility or the growth rate of offspring in the mouse model of endometriosis. Taken together, we identify hypoxia/LATS1/YAP1 as a novel pathway for the pathogenesis of endometriosis and demonstrate that targeting YAP1 might be an alternative approach to treat endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

YAP1 regulates ABCG2 and cancer cell side population in human lung cancer cells.

A small population of cancer cells called cancer-initiating cells or cancer stem cells (CSCs) are involved in drug resistance, metastasis, and cancer relapse. Finding pathways that regulate CSC is very important for clinical therapy. ATP-binding cassette sub-family G member 2 (ABCG2) plays a role in side population (SP) cell formation and contributes to chemotherapy resistance in common forms of cancer. Yes-associated protein 1 (YAP1) is a major transcriptional effector of the Hippo pathway, which plays important roles in organ size control and tumorigenesis. In this study, we found ABCG2 and YAP1 were both overexpressed in lung cancer SP cells. Disruption of YAP1 expression by siRNA attenuated the expression of ABCG2 transcript and significantly reduced the percentage of SP cells and sphere formation in lung cancer cells. Overexpression of YAP1 in lung cancers led to an increase in ABCG2 expression and increased the percentage of SP cells. However, overexpression of YAP1 in purified non-SP cells did not increase ABCG2 expression and the percentage of SP cells, which may be due to the inhibition of YAP activity through phosphorylation. YAP1 directly transcriptionally regulated ABCG2 by binding to the promoter of ABCG2. Moreover, the YAP1 inhibitor verteporfin and YAP1 siRNA downregulated ABCG2 level through inhibition of YAP1 in lung cancer cells and sensitized them to the chemotherapy drug doxorubicin. Our study adds a new function for YAP1 that may be relevant to drug resistance and cancer therapy through regulation of ABCG2 and side population cell formation in lung cancer.