Matrix Mechanotransduction via Yes-Associated Protein in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state. Methods: LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM). Results: GLC cells show significantly (P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment. Conclusions: These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.

Long non-coding TUG1 accelerates prostate cancer progression through regulating miR-128-3p/YES1 axis.

OBJECTIVE: Dysregulation of long non-coding RNAs (lncRNAs) is being found to have relevance to human cancers, including prostate cancer (PCa). Taurine-upregulated gene 1 (TUG1) has been demonstrated to have a potential oncogenic role in PCa. Then the aim of this study was to investigate the molecular mechanisms of TUG1 on PCa progression. PATIENTS AND METHODS: The expression levels of TUG1, YES proto-oncogene 1 (YES1) mRNA and miR-128-3p were assessed using quantitative real-time polymerase chain reaction. Cell proliferation ability, apoptosis, and migration and invasion capacities were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry and transwell assay, respectively. Western blot analysis was employed to evaluate the indicated proteins levels. The interaction between miR-128-3p and TUG1 or YES1 was determined using the Dual-Luciferase reporter assay. In vivo assay was used to observe the effect of TUG1 on tumor growth in vivo. RESULTS: Our data indicated that TUG1 was upregulated in PCa tissues and cells and predicted poor prognosis. TUG1 knockdown weakened PCa cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and accelerated cell apoptosis in vitro. Mechanistically, TUG1 directly interacted with miR-128-3p and miR-128-3p mediated the regulatory effects of TUG1 depletion on PCa cell progression. YES1 was a direct target of miR-128-3p and TUG1 modulated YES1 expression by sponging miR-128-3p. Moreover, TUG1 silencing repressed PCa cell progression in vitro through YES1. Additionally, TUG1 silencing mitigated tumor growth in vivo. CONCLUSIONS: Our study suggested that TUG1 silencing retarded PCa cell progression in vitro and tumor growth in vivo through miR-128-3p/YES1 axis, showing that targeting TUG1 might be a novel therapeutic strategy for PCa management.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Long noncoding RNA H19 competitively binds miR-17-5p to regulate YES1 expression in thyroid cancer.

The long noncoding RNA H19 is overexpressed in many cancers and acts as an oncogene. Here, we investigated the role of H19 in thyroid carcinogenesis and its relation to microRNA miR-17-5p and its target gene YES1. H19 expression was higher in tumor samples and in thyroid cancer cell lines than nontumor tissues and normal thyroid cells. H19 knockdown and ectopic expression in the TPC-1 and NIM thyroid cancer cell lines showed that overexpression of H19 promoted proliferation, migration, and invasion, whereas H19 knockdown reduced cell viability and invasion and induced growth arrest in vitro and in vivo. H19 was identified as a target of miR-17-5p, by Dual-Luciferase Reporter assays and RNA-binding protein immunoprecipitation assays. H19 antagonized the function of miR-17-5p on upregulation of its target YES1 and inhibited miR-17-5p-induced cell cycle progression. Our results suggest that H19 functions as a competitive endogenous RNA (ceRNA) by acting as a sink for miR-17-5p, revealing a potential ceRNA regulatory network involving H19 and miR-17-5p with a role in the modulation of YES1 expression. This mechanism may contribute to a better understanding of thyroid cancer pathogenesis and provide new insights into the treatment of this disease.

miR-203 downregulates Yes-1 and suppresses oncogenic activity in human oral cancer cells.

The purpose of this study was to elucidate the molecular mechanisms of microRNA-203 (miR-203) as a tumor suppressor in KB human oral cancer cells. MicroRNA microarray results showed that the expression of miR-203 was significantly down-regulated in KB cells compared with normal human oral keratinocytes. The viability of KB cells was decreased by miR-203 in the time- and dose-dependent manners. In addition, over-expressed miR-203 not only increased the nuclear condensation but also significantly increased the apoptotic population of KB cells. These results indicated that the over-expression of miR-203 induced apoptosis of KB cells. Furthermore, the target gene array analyses revealed that the expression of Yes-1, a member of the Src family kinases (SFKs), was significantly down-regulated by miR-203 in KB cells. Moreover, both the mRNA and protein levels of Yes-1 were strongly reduced in KB cells transfected with miR-203. Therefore, these results indicated that Yes-1 is predicted to be a potential target gene of miR-203. Through a luciferase activity assay, miR-203 was confirmed to directly targets the Yes-1 3' untranslated region (UTR) to suppress gene expression. Therefore, our findings indicate that miR-203 induces the apoptosis of KB cells by directly targeting Yes-1, suggesting its application in anti-cancer therapeutics.