Targeting Hippo/YAP in intrahepatic cholangiocarcinoma: Promising molecules in cancer therapy.

The tumorigenesis of intrahepatic cholangiocarcinoma (ICC) has been identified to be exceptionally involved in dysregulated Hippo/Yes-associated protein (YAP) signaling pathway (Hippo/YAP). Hippo/YAP functions as a master regulator engaged in a plethora of physiological and oncogenic processes as well. Therefore, the aberrant Hippo/YAP could serve as an Achilles' heel regarding the molecular therapeutic avenues for ICC patients. Herein, we comprehensively review the recent studies about the underlying mechanism of disrupted Hippo/YAP in ICC, how diagnostic values could be utilized upon the critical genes in this pathway, and what opportunities could be given upon this target pathway.

SEPT6 drives hepatocellular carcinoma cell proliferation, migration and invasion via the Hippo/YAP signaling pathway.

Septin 6 (SEPT6) is a member of the GTP­binding protein family that is highly conserved in eukaryotes and regulates various biological functions, including filament dynamics, cytokinesis and cell migration. However, the functional importance of SEPT6 in hepatocellular carcinoma (HCC) is not completely understood. The present study aimed to investigate the expression levels and roles of SEPT6 in HCC, as well as the underlying mechanisms. The reverse transcription quantitative PCR, western blotting and immunohistochemistry staining results demonstrated that SEPT6 expression was significantly elevated in HCC tissues compared with corresponding adjacent non­tumor tissues, which indicated that SEPT6 expression may serve as a marker of poor prognosis for HCC. By performing plasmid transfection and G418 treatment, stable SEPT6­knockdown and SEPT6­overexpression cell lines were established. The Cell Counting Kit­8, flow cytometry and Transwell assay results demonstrated that SEPT6 overexpression significantly increased HCC cell proliferation, cell cycle transition, migration and invasion compared with the Vector group, whereas SEPT6 knockdown displayed significant suppressive effects on HCC cell lines in vitro compared with the control group. Mechanistically, SEPT6 might facilitate F­actin formation, which induced large tumor suppressor kinase 1 dephosphorylation, inhibited Hippo signaling, upregulated yes­associated protein (YAP) expression and nuclear translocation, and upregulated cyclin D1 and matrix metallopeptidase 2 (MMP2) expression. Furthermore, YAP overexpression significantly reversed SEPT6 knockdown­induced inhibitory effects on HCC, whereas YAP knockdown significantly inhibited the oncogenic effect of SEPT6 overexpression on HCC. Collectively, the present study demonstrated that SEPT6 may promote HCC progression by enhancing YAP activation, suggesting that targeting SEPT6 may serve as a novel therapeutic strategy for HCC.

MicroRNA­93 promotes angiogenesis and attenuates remodeling via inactivation of the Hippo/Yap pathway by targeting Lats2 after myocardial infarctionω.

Inactivation of the Hippo pathway protects the myocardium from cardiac ischemic injury. MicroRNAs (miRs) have been reported to play pivotal roles in the progression of myocardial infarction (MI). The present study examined whether miR­93 could promote angiogenesis and attenuate remodeling after MI via inactivation of the Hippo/Yes­associated protein (Yap) pathway, by targeting large tumor suppressor kinase 2 (Lats2). It was identified that transfection of human umbilical vein endothelial cells with miR­93 mimic significantly decreased Lats2 expression and Yap phosphorylation, increased cell viability and migration, and attenuated cell apoptosis following hypoxia/reoxygenation injury. Moreover, increased expression of miR­93 resulted in an improvement of cardiac function, promotion of angiogenesis and attenuation of remodeling after MI. Additionally, miR­93 overexpression significantly decreased intracellular adhesion molecule 1 and vascular cell adhesion protein 1 expression levels, as well as attenuated the infiltration of neutrophils and macrophages into the myocardium after MI. Furthermore, it was found that miR­93 overexpression significantly suppressed Lats2 expression and decreased the levels of phosphorylated Yap in the myocardium after MI. Collectively, the present results suggested that miR­93 may exert a protective effect against MI via inactivation of the Hippo/Yap pathway by targeting Lats2.

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.

Downregulation of YAP inhibits proliferation and induces apoptosis in Eca-109 cells.

A previous study reported that Yes-associated protein (YAP) gene was overexpressed in esophageal squamous cell carcinoma (ESCC); however, the exact role of YAP in ESCC remains largely unclear. The present study aimed to investigate the effects of YAP inhibition on ESCC. In order to investigate the exact role of YAP in ESCC cells, a stable YAP low-expression ESCC cell line was established using YAP-small interfering RNA. MTT assay was performed to examine the cell proliferation ability, while flow cytometry were used to detect the cell apoptosis and cell cycle distribution. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were applied for mRNA and protein level detection, respectively. The results suggested that YAP gene inhibition significantly repressed the ECA-109 cell proliferation and induced cell apoptosis, whereas this inhibition had no significant effects on cell cycle. Furthermore, the expression levels of cell apoptosis-associated proteins were determined in the current study, and the data demonstrated that the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein ratio and phosphorylated extracellular signal-regulated kinase expression were significantly reduced, while the p53 and caspase 3 levels were notably increased in YAP gene-inhibited ECA-109 cells. In conclusion, the current study revealed that YAP gene inhibition suppresses the proliferation and induces apoptosis in ECA-109 cells, indicating that the YAP gene serves as an oncogene in ESCC.

Furin inhibitor D6R suppresses epithelial-mesenchymal transition in SW1990 and PaTu8988 cells via the Hippo-YAP signaling pathway.

Hexa-D-arginine (D6R), an inhibitor of furin, has potential therapeutic applications in different types of human tumor. However, the function of D6R in targeting pancreatic cancer cells remains to be elucidated. In the present study, the proliferation, invasion and migration abilities of SW1990 and PaTu8988 cells were examined using a Cell Counting Kit-8, and Transwell and wound healing assays. Subsequently, the expression of proteins associated with epithelial-mesenchymal transition (EMT) and the Hippo-yes-associated protein (YAP) pathway were detected using western blot analysis. It was revealed that D6R significantly inhibited the proliferation, migration and invasion abilities of SW1990 and PaTu8988 cells. Additionally, D6R led to the upregulation of E-cadherin (an epithelial marker), and the downregulation of N-cadherin and vimentin (mesenchymal markers) in SW1990 and PaTu8988 cells. Furthermore, the results of the present study revealed that D6R significantly affected the YAP phosphorylation level and the total YAP protein level, indicating that D6R was functionally involved in the Hippo-YAP signaling pathway. It has been suggested that D6R-suppressed EMT in SW1990 and PaTu8988 cells may occur via the Hippo-YAP pathway and that it may be a feasible drug to ameliorate the malignant phenotype of SW1990 and PaTu8988 cells.

Liraglutide suppresses proliferation and induces adipogenic differentiation of 3T3-L1 cells via the Hippo-YAP signaling pathway.

Liraglutide, as a glucagon-like peptide­1 analogue, is used to treat type 2 diabetes mellitus and obesity. Previous findings have demonstrated the effects of liraglutide on adipogenesis; however, the underlying mechanism involved in this process remains to be elucidated. In the present study, to certify the effect of liraglutide on adipogenesis and explore the possible underlying mechanism involved in this process, preadipocyte 3T3­L1 cells were cultured in adipocyte­inducing medium and treated with liraglutide. Subsequently, the expression levels of the master transcription factors and adipocyte­specific genes were measured by reverse transcription­quantitative polymerase chain reaction and immunoblotting analysis. Lipid droplet production was detected by Oil red O staining. Cell proliferation was determined by a Cell Counting Kit-8 assay and cell immunofluorescence for Ki67, and apoptosis was assessed by flow cytometry. Next, the expression levels of the core components in the Hippo­yes­associated protein (YAP) signaling pathway as well as YAP­specific target genes were measured. Finally, short interfering RNAs of mammalian ste20 kinase 1/2 (MST1/2), a key protein kinase in the Hippo­YAP pathway, were used to determine whether liraglutide regulated adipogenic differentiation via the Hippo­YAP pathway. It was demonstrated that liraglutide promoted adipogenic differentiation, suppressed proliferation, did not affect apoptosis of 3T3­L1 cells and activated the Hippo­YAP signaling pathway at the initial stage of adipogenesis. Silencing of MST1 counteracted the effect of increasing adipogenesis by liraglutide. These results suggested that liraglutide may activate the Hippo­YAP signaling pathway leading to the inhibition of proliferation of preadipocyte 3T3­L1 cells, and result in cells achieving transformation into mature adipocytes sooner. Taken together, the results of the present study may expand knowledge of the underlying mechanism of liraglutide facilitating adipogenesis, and may contribute to the development of GLP­1 receptor agonists for weight loss and increased insulin sensitivity.