Large Tumor Suppressor Research Articles

SHCBP1 promotes tumor cell proliferation, migration, and invasion, and is associated with poor prostate cancer prognosis.

Prostate cancer (PCa) is an aggressive tumor. SHC SH2-domain-binding protein 1 (SHCBP1) has been identified frequently upregulated in various cancers, in addition to PCa. The aims of this study were to determine the relationships between SHCBP1 and clinicopathological characteristics of PCa and to explore the role of SHCBP1 in PCa proliferation and progression. Tissue microarray and immunohistochemistry were used to determine the prognostic significance of SHCBP1. The relationship between clinicopathological characteristics of PCa and SHCBP1 was then analyzed using Cox regression analyses. To investigate SHCBP1 functions in vitro and in vivo, we knocked down SHCBP1 in PCa cell lines and established xenograft mice models. A series of cytological function assays were utilized to determine the role of SHCBP1 in cell proliferation, migration, invasion, and apoptosis. SHCBP1 was significantly upregulated in PCa tissues compared with BPH tissues. Patients with a higher expression of SHCBP1 were associated with poor survival outcomes than those with a lower expression of SHCBP1. Lentivirus-mediated shRNA knockdown of SHCBP1 in prostate cancer cell lines diminished cell growth, migration, and invasion dramatically both in vitro and in vivo, accompanied by an enhanced expression of large tumor suppressor 1 (LATS1) and tumor protein P53 (TP53) and inhibition of MDM2 proto-oncogene (MDM2), which suggested that SHCBP1 may promote proliferation and invasion in vitro via the LATS1-MDM2-TP53 pathway. The results of cycloheximide (CHX) and MG-132 assays indicated that SHCBP1 knockdown could attenuate the degradation of TP53 by the proteasome, prolong the half-life of TP53, and enhance the stabilization of TP53. These findings suggest that SHCBP1 overexpression contributes to PCa progression and that targeting SHCBP1 might be therapeutically beneficial to patients with PCa.

Association analysis between SNPs in LATS1 and LATS2 and non-cardia gastric cancer

BackgroundMany studies have found that large tumor suppressor kinase 1 (LATS1) and LATS2 play important roles in many diseases, but studies have been rare on the relationship between these genes and non-cardia gastric cancer (GC). We performed a case-control association study to investigate the associations between single nucleotide polymorphisms (SNPs) in LATS1 and LATS2 genes and Helicobacter pylori (H. pylori) infection as well as the risk of non-cardia GC.MethodsFirst, H. pylori infection was determined by the serological test using enzyme-linked immunoassay. Then genotyping of SNPs was performed for 808 samples by the Taqman method. Finally, unconditional logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs), adjusted for age and gender, for the association of each SNP with the infection of H. pylori, the risk of non-cardia gastric cancer, as well as the expression of LATS1 and LATS2 proteins in non-cardia GC tissues, using the codominant, dominant, recessive, overdominant, and log-additive inheritance models, respectively.ResultsThe statistical results showed that LATS2 rs9552315 was associated with H. pylori infection, and the CC + CT genotype could reduce the risk of H. pylori infection (odds ratio [OR]: 0.549, 95% confidence interval [CI]: 0.339–0.881, P < 0.05) compared with the TT genotype in a dominant model. LATS1 rs9393175 was associated with the risk of non-cardia GC, and the AG genotype reduced the risk of non-cardia GC (OR: 0.702, 95% CI: 0.516–0.952, P < 0.05) compared with the GG + AA genotype in an overdominant model. LATS2 rs9509492 was associated with the risk of GC in an log-additive model. No associations were found between five SNPs and expression of LATS1 and LATS2 proteins in non-cardia GC tissue.ConclusionsLATS2 rs9552315 CT genotype may be a protective factor against infection of H. pylori. LATS1 rs9393175 AG genotype and LATS2 rs9509492 GG genotype may be protective factors for non-cardia GC.

Circular RNA circ_0001368 inhibited growth and invasion in renal cell carcinoma by sponging miR-492 and targeting LATS2

Circular RNAs (circRNAs) act as essential regulators in the tumorigenesis of renal cell carcinoma. Our study aims to investigate the underlying function and the molecular mechanisms of circ_0001368 in renal cell carcinoma. The qRT-PCR results indicate that the circ_0001368 expression level was downregulated in the carcinoma cells and tissues of the renal cell. circ_0001368 weakened cell proliferation and invasion in the ACHN and 786-O cells. The luciferase reporter assay showed that circ_0001368 functioned as an endogenous sponge for miR-492. The transwell and CCK8 assays showed that circ_0001368 suppressed cell proliferation and invasion in the ACHN and 786-O cells. Large tumor suppressor kinase 2 (LATS2) was confirmed by Targetscan as a target gene of miR-492. The overexpression of LATS2 repressed the growth and invasion of ACHN and 786-O cells. circ_0001368 upregulated the LATS2 expression and suppressed ACHN and 786-O cell growth and invasion by sponging miR-492. circ_0001368 suppressed the proliferation ability of 786-O in vivo. In conclusion, circ_0001368 was identified in this study as a novel anti-tumor RNA in renal cell carcinoma and can function as a potential therapeutic target for renal cell carcinoma treatment.

Upregulation of miR-215 attenuates propofol-induced apoptosis and oxidative stress in developing neurons by targeting LATS2

BackgroundPropofol is an intravenous anesthetic agent that commonly induces significant neuroapoptosis. MicroRNAs (miRNAs) have been reported to participate in the regulation of propofol exposure-mediated neurotoxicity. MiR-215, as one of miRNAs, was found to regulate nerve cell survival. However, the mechanism through which miRNAs regulate propofol exposure-mediated neurotoxicity is still unclear.MethodsReal-time PCR was used to detect miR-215 expression level. Cell viability was measured using MTT assay. Cell apoptosis was examined via flow cytometry analysis. ROS, MDA, LDH and SOD levels were assayed through ELISA kits. Dual luciferase reporter assay identified the interaction between miR-215 and large tumor suppressor 2 (LATS2). Protein level was detected using western blot analysis.ResultsMiR-215 expression was downregulated in propofol-treated rat hippocampal neurons. MiR-215 mimics promoted cell viability and reduced apoptosis in propofol-treated neonatal rat hippocampal neuron. MiR-215 mimics also caused inhibition of oxidative stress as evidenced by suppression of ROS, MDA and LDH levels as well as increase of SOD level. In addition, we found that large tumor suppressor 2 (LATS2) is a target of miR-215 and miR-215 mimics decreased LATS2 level in propofol-treated neonatal rat hippocampal neuron. Further, LATS2 overexpression suppressed the effect of miR-215 on propofol-induced apoptosis and oxidative stress in neonatal rat hippocampal neuron.ConclusionTaken together, we demonstrate that miR-215 attenuates propofol-induced apoptosis and oxidative stress in neonatal rat hippocampal neuron by targeting LATS2, suggesting that miR-215 may provide a new candidate for the treatment of propofol exposure-induced neurotoxicity.

Promoter Hypermethylation of LATS2 Gene in Oral Squamous Cell Carcinoma (OSCC) Among North Indian Population.

Large Tumor Suppressor (LATS2) gene are Tumor Suppressor gene, linked with epigenetic modifications. LATS2 promoter hypermethylation is an important epigenetic silencing mechanism leading to cancer. Cancer is the most common, vicious and dangerously increasing diseases of the world today, associated with high morbidity and mortality. Oral cancers (OC) are the blazing universal dilemma and is the sixth most frequent cancer observed in Indian population. Tobacco consumption is the main cause of the increase in OSCC. The association between LATS2 in the pathogenesis of cancers propose that their combination might be studied as a possible molecular marker for particular subgroups of patients. Therefore, the present study tried to investigate whether LATS2 promoter methylation was associated with oral squamous cell carcinoma (OSCC) in North Indian subjects. DNA methylation quantitative studies of LATS2 Tumor Suppressor genes were performed by methylation-specific polymerase chain reaction (MSP). 38 out of 70 patients (55 %) were found to be methylated for LATS2 gene, a statistically significant result was obtained (p-value < 0.005) for LATS2 genes. The results suggest that epigenetic changes may be related to the down-regulation of LATS2 expression. It can be concluded that LATS2 gene plays a significant role in the diagnosis of cancer and provide a better alternative as a diagnostic biomarker. Our data infer that a low LATS2 expression due to methylation may contribute to the cancer progression and could be useful for the diagnosis of OSCC. Therefore, investigation of promoter methylation in such genes may provide a biomarker which may prove to be useful in early detection of Oral Cancer.

Knockout of LATS1 induces neoplastic phenotype in hepatic oval cells.

BackgroundPrimary liver cancer (PLC) is the second leading cause of cancer-related death worldwide. It has been reported that PLC can be originated from malignant transformed adult hepatic progenitor cells. Mammalian large tumor suppressor kinase 1 (LATS1) is one of the core components of the Hippo pathway and it has been implicated in regulating invasion and metastasis of different cancer cell. However, the underlying connections between hepatic progenitor cells and LATS1 in the pathogenesis of PLC are still elusive.MethodsLATS1 gene knockout (LATS1-KO) hepatic oval cells (HOCs) were constructed by the CRISPR/Cas9 system. Cell viability was evaluated by the CCK-8 assay. Cell migration was measured by scrape assay. Cell invasion was examined by Transwell assay. Cell apoptosis was evaluated by flow cytometry. The expression of LATS1 and Yes-associated protein (YAP) in HOCs was determined by Q-PCR and Western blot analysis.ResultsHere, we found that knockout of LATS1 significantly induced the migration and invasion of WB-F344 cells. Knockdown of YAP suppressed the neoplastic phenotype of LATS1-KO WB-F344 cells. Furthermore, overexpression of YAP promoted the migration and invasion of LATS1-KO WB-F344 cells.ConclusionsIn summary, the current study demonstrated that LATS1 is required for inhibiting the neoplastic phenotype of normal hepatic progenitor cell via downregulating YAP.

MicroRNA‑93 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 miR-93 could promote angiogenesis and attenuate remodeling after MI via inactivation of the Hippo/Yes-associated protein (Yap) pathway, by targeting large tumor suppressor kinase 2 (Lats2). It was identified that transfection of human umbilical vein endothelial cells with miR-93 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 miR-93 resulted in an improvement of cardiac function, promotion of angiogenesis and attenuation of remodeling after MI. Additionally, miR-93 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 miR-93 overexpression significantly suppressed Lats2 expression and decreased the levels of phosphorylated Yap in the myocardium after MI. Collectively, the present results suggested that miR-93 may exert a protective effect against MI via inactivation of the Hippo/Yap pathway by targeting Lats2.

Effects of fucoidan on gut flora and tumor prevention in 1,2-dimethylhydrazine-induced colorectal carcinogenesis

Colorectal cancer (CRC) is one of the major malignancies in humans. This study was designed to evaluate the effects of fucoidan on gut flora and tumor prevention in 1,2-dimethylhydrazine-induced colorectal carcinogenesis in rats. We found that dietary fucoidan treatment decreased the tumor incidence and mean tumor weight and increased cell apoptosis. Fucoidan treatment decreased the expression of β-catenin C-Myc, CyclinD1 and Survivin, while the Hippo pathway was activated with increased phosphorylation levels of mammalian sterile 20-like kinase 1 and 2, large tumor suppressor 1 and 2, and Yes-associated protein. Compared with the model group, the levels of interleukin (IL)-17 and IL-23 were decreased, but the levels of interferon-γ, IL-4 and IL-10 were increased, in the fucoidan group. Fucoidan treatment increased natural killer cells in peripheral blood and the proportion of CD4+ T cells. Immunofluorescence detection of colorectal tumor tissues showed decreased expression of Foxp3 and up-regulated expression of CD68 in the fucoidan group. Moreover, fucoidan treatment decreased the levels of diamine oxidase and lipopolysaccharides and up-regulated the levels of tight junction proteins. 16S rDNA high-throughput sequencing revealed that fucoidan treatment decreased the abundance of Prevotella and increased the abundance of Alloprevotella. Fucoidan increased the levels of butyric acid and valeric acid compared to the model group. This study provides experimental evidence that dietary fucoidan may prevent colorectal tumorigenesis by regulating gut microecology and body immunity. Meanwhile, fucoidan activated the Hippo pathway and down-regulated the β-catenin pathway to induce tumor cell apoptosis and suppress tumor growth.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate.

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.

Structural Disorder and Induced Folding of the WWC Family Proteins

The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. Each protein contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains are named for two conserved tryptophans spaced 23 residues apart and adopt a three‐stranded antiparallel beta sheet fold. The WWC proteins are “atypical” in that, the second WW domain (WW2) contains an isoleucine rather than a conserved tryptophan. Recent studies of the KIBRA WW domains shows that this substitution leads to unfolding of the WW2 domains. However, the domain undergoes binding‐induced folding when bound to some partners but remains largely unfolded when bound to others. High sequence similarity of the WW domains in the WWC family suggests that unfolding and binding induced folding of the WW2 domain may be conserved in this family, but it is unclear why some partners induce folding of the WW2 domain while others do not. Our working hypothesis is that partners which contain PPxY motifs in close proximity will bind the WWC proteins with relatively high affinity and induce folding of the WW2 domain. In this study, we test this hypothesis with two binding partners, Large Tumor Suppressor 1 (LATS1), a kinase and tumor suppressor, and Synaptopodin (SYNPO), a synaptic and podocyte protein. We demonstrate by isothermal titration calorimetry that wildtype LATS1 and SYNPO which have two PPxY motifs each, separated by 194 and 15 residues respectively, bound KIBRA and WWC3 with modest micromolar binding affinities. However, mutant LATS1 (LATS1‐VA) and SYNPO (SYNPO_AA) constructs in which the PPxY motifs are separated by two amino acids, bound KIBRA and WWC3 with 20 to 180‐fold enhanced affinity. Using solution magnetic resonance spectroscopy, we demonstrated that the WW2 domain of KIBRA and WWC3 remain unfolded when bound to wildtype LATS1 and SYNPO, while the enhanced binding of the mutants LATS1_VA and SYNPO_AA is accompanied by folding of the WW2 domain. The structural heterogeneity of the WW2 domain may be an important for modulating binding to diverse PPxY motif partners.Support or Funding InformationThis work is supported by start‐up funds from Oregon State University (OSU), OSU College of Science Disease Mechanism and Prevention Fund, and a New Investigator Award from the Medical Research Foundation of Oregon. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162

Expression of large tumour suppressor (LATS) kinases modulates chemotherapy response in advanced non-small cell lung cancer.

BackgroundThe Hippo signalling pathway plays an important role in regulating organ size and cell proliferation. Down-regulation of large tumour suppressor (LATS) protein homologs LATS1 or LATS2 has been found in lung cancer. LATS1 and LATS2 are the core components of the Hippo signalling pathway. LATS1 and LATS2 share some conserved structural features and exhibit redundant biological functions. The aim of this study was to dissect the interaction between these two homologs.MethodsIn lung adenocarcinoma (AD) cells, protein expression of LATS1 and LATS2 were determined by western blotting; cell viability and apoptosis were measured by MTT and annexin V staining after treatment with cisplatin; subcellular distributions of LATS proteins were determined by immunofluorescence microscopy; LATS2 expression was modulated by shRNA-mediated knockdown or ectopic expression in cancer cell lines.ResultsManipulation of the expression of these two LATS kinases influenced cisplatin response in advanced lung AD cell lines. High LATS2-to-LATS1 ratio in H2023 cells was associated with cisplatin resistance, while low LATS2-to-LATS1 ratio in CL1-0 and CL83 cells was associated with sensitivity to cisplatin. Manipulating the LATS2-to-LATS1 ratio by LATS2 over-expression in CL1-0 and CL83 rendered them resistant to cisplatin treatment, whereas LATS2 knockdown in H2023 alleviated the LATS2-to-LATS1 ratio and sensitized cancer cells to cisplatin exposure.ConclusionsOur data suggested that the ratio of expression of LATS kinases played a role in the modulation of cisplatin sensitivity in advanced lung AD, and targeting of LATS proteins as a novel therapeutic strategy for lung AD deserves further investigation.

MP08-20 KIBRA AND PHOSPHORYLATED LATS-2 ARE NEGATIVELY ASSOCIATED WITH MALIGNANT AGGRESSIVENESS AND ARE PROGNOSTIC INDICATORS IN PATIENTS WITH RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVE:The phosphorylation of large tumor suppressor (LATS)-1/2 inactivates Yes-associated protein (YAP) and results in the inhibition of malignant aggressiveness in several can...

Stretch‐induced activation of Hippo signaling in lung microvascular endothelial cells – a novel mechanism of overventilation‐induced pulmonary fibrosis

Mechanical ventilation is a mainstay of current therapy for patients with acute respiratory distress syndrome (ARDS). Yet despite its benefits, mechanical ventilation can also cause ventilator induced lung injury (VILI) that may ultimately progress to pulmonary fibrosis. We aimed to elucidate cellular mechanisms by which mechanical ventilation triggers pulmonary fibrosis. Specifically, we focused on the mechanosensitive transcriptional co‐factor TAZ, a key player in the HIPPO‐signaling pathway involved in mechanosensation. In the HIPPO pathway, phosphorylation of Large Tumor Suppressor Kinases (LATS) regulates the phosphorylation state of TAZ and its translocation to the nucleus. Despite its emerging relevance in tissue fibrosis, TAZ activation, its underlying mechanisms and downstream profibrotic effects upon mechanical overventilation or its in vitro correlate cyclic stretch have not been elucidated.ObjectivesTo examine the effect of mechanical stretch on the nuclear translocation of TAZ in endothelial cells, and to identify underlying regulatory mechanisms effects both in vitro and in vivo.MethodsIn vitro: human pulmonary microvascular endothelial cells (HPMEC) were subjected to stretch regimens at 5%, mimicking low tidal volume ventilation, or 18% mimicking high tidal volume ventilation at 0.25 Hz for 1, 3, 6, or 12h on a Flexcell Tension System and compared to static control. In vivo: To mimic ARDS in mice, Endothelial TAZ‐deficient and corresponding control were challenged with HCl (pH 1.0; 3 mL/kg), recovered for 3 days, ventilated for 2 hours at VT of 20ml/kg and PEEP of 2 cmH2O; and compared to unventilated controls. Evidence of fibrosis was detected histologically two weeks after ventilation.ResultsIn vitro: Mechanical stretch induced the translocation of TAZ from the cytosol to the nucleus in a dose‐ and time‐dependent manner. Specifically, the nuclear to cytoplasmic ratio of TAZ increased after 3h of stretch at 18% compared to the control whereas stretching cells at 5% increased TAZ nuclear localization after 6h. Stretch‐induced TAZ translocation was blocked by the inhibition of Aurora kinases with SNS 314 mesylate (10 μMol/L). Consistently, inhibition of Aurora kinases decreased LATS2 phosphorylation at Ser83, a modulatory site of its activity. In vivo: Histological analysis of lung tissue using Masson’s trichrome and H&amp;E stain showed marked peribronchiolar and perivascular collagen deposition as well as bronchial wall thickening due to extensive infiltration of inflammatory cells in ventilated control, yet not in endothelial TAZ‐deficient mice.ConclusionThese data suggest that mechanical stretch activates TAZ nuclear translocation, at least in part, via an Aurora kinase and LATS2‐dependent mechanism. Knocking down TAZ in endothelial cells protected mice from developing pulmonary fibrosis following HCL‐overventilation. Interventions targeted at the Hippo‐signaling pathway and its upstream regulation may present potential strategies to counteract the detrimental effects of mechanical overventilation.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research.

Sitagliptin affects gastric cancer cells proliferation by suppressing Melanoma-associated antigen-A3 expression through Yes-associated protein inactivation.

Sitagliptin is an emerging oral hypoglycemic agent that inhibits the development of a wide variety of tumors. Current researches indicate that the abnormal activation of Yes‐associated protein (YAP) promotes the proliferation and poor prognosis of multiple tumors. However, the ability of sitagliptin to regulate YAP and its effects on gastric cancer (GC) cells remain unclear. Here, we first showed that sitagliptin inhibited the proliferation of GC cells, and this inhibition was regulated by Hippo pathway. Sitagliptin phosphorylated YAP in a large tumor suppressor homolog‐dependent manner, thereby inhibiting YAP nuclear translocation, and promoted YAP cytoplasm retention. This inhibition can be blocked by adenosine 5′‐monophosphate‐activated protein kinase (AMPK). Moreover, sitagliptin could reduce the expression of tumor‐testis antigen Melanoma‐associated antigen‐A3 through YAP. In conclusion, sitagliptin may have a potential inhibitory effect on GC by AMPK/YAP/melanoma‐associated antigen‐A3 pathway.

The Emerging Roles of NDR1/2 in Infection and Inflammation.

The nuclear Dbf2-related (NDR) kinases NDR1 and NDR2 belong to the NDR/LATS (large tumor suppressor) subfamily in the Hippo signaling pathway. They are highly conserved from yeast to humans. It is well-known that NDR1/2 control important cellular processes, such as morphological changes, centrosome duplication, cell proliferation, and apoptosis. Recent studies revealed that NDR1/2 also play important roles in the regulation of infection and inflammation. In this review, we summarized the roles of NDR1/2 in the modulation of inflammation induced by cytokines and innate immune response against the infection of bacteria and viruses, emphasizing on how NDR1/2 regulate signaling transduction through Hippo pathway-dependent and -independent manners.

Downregulation of microRNA-224-3p Hampers Retinoblastoma Progression via Activation of the Hippo-YAP Signaling Pathway by Increasing LATS2.

PurposeThe pivotal role of microRNAs (miRNAs or miRs) has been proved in the pathogenesis of retinoblastoma. miR-224-3p is demonstrated to be involved in several tumors. However, the underlying mechanism of miR-224-3p in retinoblastoma is yet to be investigated. Therefore, this study was designed to identify the regulation of miR-224-3p in human retinoblastoma.MethodsThe expression pattern of miR-224-3p and large tumor suppressor 2 (LATS2) in retinoblastoma was measured by reverse transcription quantitative polymerase chain reaction. Afterward, the interaction between miR-224-3p and LATS2 was identified using a dual luciferase reporter gene assay. Next, gain-of-function and loss-of-function approaches were employed to examine the effects of miR-224-3p and LATS2 as well as their interaction on cell apoptosis, proliferation and angiogenesis abilities, and tumorigenesis. Whether the Hippo-YAP signaling pathway was involved in tumorigenesis was analyzed by determining downstream genes.ResultsLATS2 was downregulated in retinoblastoma, and its overexpression promoted apoptosis and suppressed proliferation of retinoblastoma cells. miR-224-3p, highly expressed in retinoblastoma, inhibited the expression of its target gene LATS2, which inhibited activation of the Hippo-YAP signaling pathway. Suppression of miR-224-3p promoted apoptosis while suppressing the proliferation of retinoblastoma cells and angiogenesis. Tumor progression induced by upregulation of miR-224-3p was diminished by restoration of LATS2. It was observed that tumor growth and angiogenesis were reduced by depleted miR-224-3p in the animal experiments.ConclusionsThe present study suggests that miR-224-3p targets LATS2 and blocks the Hippo-YAP signaling pathway activation, thus preventing the progression of retinoblastoma, which could be a new therapeutic target for retinoblastoma.

Effect of Downregulating the Hippo Pathway Members YAP1 and LATS2 Transcripts on Early Development and Gene Expression Involved in Differentiation in Porcine Embryos.

In mouse development, differentiation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to blastocyst stage is regulated by the Hippo pathway; however, the functions of the Hippo pathway in porcine embryogenesis have not been investigated. In the present study, we examined the gene expression patterns of the Hippo pathway members yes-associated protein 1 (YAP1) and large tumor suppressor 2 (LATS2) and the functions of these genes during porcine preimplantation development using RNA interference. Both YAP1 and LATS2 mRNA levels were shown high in the in vitro matured oocytes and 1-cell stage embryos and fell progressively with development. YAP1 nuclear localization was detected at the morula and blastocyst stages. Downregulation of either YAP1 or LATS2 inhibited porcine preimplantation development and affected the expression levels of POU class 5 homeobox 1 (OCT-4) and SRY-related HMG-box gene 2 (SOX2), transcription factors necessary for the ICM/TE differentiation. Taken together, YAP1 and LATS2 are essential for porcine preimplantation development, and it is possible that the Hippo pathway has important roles in porcine ICM/TE segregation.

Furry protein suppresses nuclear localization of yes-associated protein (YAP) by activating NDR kinase and binding to YAP

The Hippo signaling pathway suppresses cell proliferation and tumorigenesis. In the canonical Hippo pathway, large tumor suppressor kinases 1/2 (LATS1/2) phosphorylate the transcriptional coactivator yes-associated protein (YAP) and thereby suppress its nuclear localization and co-transcriptional activity. Nuclear Dbf2-related kinases 1/2 (NDR1/2), which are closely related to LATS1/2, also phosphorylate and inactivate YAP by suppressing its nuclear localization. Furry (FRY) is a cytoplasmic protein that associates with NDR1/2 and activates them, but its role in the nuclear/cytoplasmic localization of YAP remains unknown. Here, we constructed FRY-knockout cell lines to examine the role of FRY in YAP's cytoplasmic localization. FRY depletion markedly increased YAP nuclear localization and decreased NDR1/2 kinase activity and YAP phosphorylation levels, but did not affect LATS1/2 kinase activity. This indicated that FRY suppresses YAP's nuclear localization by promoting its phosphorylation via NDR1/2 activation. NDR1/2 depletion also promoted YAP nuclear localization, but depletion of both FRY and NDR1/2 increased the number of cells with YAP nuclear localization more strongly than did depletion of NDR1/2 alone, suggesting that FRY suppresses YAP nuclear localization by a mechanism in addition to NDR1/2 activation. Co-precipitation assays revealed that Fry uses its N-terminal 1-2400-amino-acid-long region to bind to YAP. Expression of full-length FRY or its 1-2400 N-terminal fragment restored YAP cytoplasmic localization in FRY-knockout cells. Taken together, these results suggest that FRY plays a crucial role in YAP cytoplasmic retention by promoting YAP phosphorylation via NDR1/2 kinase activation and by binding to YAP, leading to its cytoplasmic sequestration.

Experimental study of human amniotic mesenchymal stem cell exosome promoting fibroblasts migration through microRNA-135a

To investigate the effect of microRNA-135a (miR-135a) in human amnion mesenchymal stem cell exosome (hAMSC-Exo) on the migration of fibroblasts. The hAMSC-Exo was extracted with exosomes separation kit and identified, the effect of hAMSC-Exo on fibroblasts migration was detected by scratch test. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-135a gene in hAMSC-Exo after overexpression of miR-135a. Scratch test was used to detect the effect of hAMSC-Exo on the migration of fibroblasts after overexpression and knockdown of miR-135a. Western blot was used to detect the migration related proteins of fibroblasts [large tumor suppressor 2 (LATS2), E-cadherin, N-cadherin, and α smooth muscle actin (α-SMA)] after overexpression and knockdown of miR-135a. The 293T cell exosomes and hAMSC-Exo were used as control. hAMSC-Exos were extracted successfully. Scratch test results showed that hAMSC group had the strongest ability to promote fibroblasts migration, and GW4869 (exosome inhibitor) treatment group had reduced ability to promote fibroblasts migration. qRT-PCR test showed that the relative expression of miR-135a gene in hAMSC-Exo increased significantly after over expression of miR-135a. Scratch test results showed that after over expression of miR-135a, hAMSC-Exo enhanced the migration ability of fibroblasts, while after knockdown of miR-135a, hAMSC-Exo weakened the migration ability of fibroblasts. Western blot results showed that the expressions of E-cadherin, N-cadherin, LATS2 were down regulated and α-SMA was up regulated in each hAMSC-Exo treatment group when compared with 293T cell exosomes group; after over expression of miR-135a, hAMSC-Exo decreased the expressions of E-cadherin, N-cadherin, LATS2 and increased the expression of α-SMA; while after knockdown of miR-135a, the ability of hAMSC-Exo was weakened. miR-135a in hAMSC-Exo can promote fibroblasts' migration, inhibit the expressions of E-cadherin, N-cadherin, LATS2, and promote the expression of α-SMA.

Selectivity and Specificity in WW Domain-PPxY Interactions

WW domain-PPxY (where x is any amino acid) interactions play multiple roles in regulating cell growth. A puzzling aspect of these interactions is how the same PPxY motif protein selects between diverse WW domain proteins. Using recombinant PPxY motif proteins Angiomotin-Like 1 (AMOTL1) and Large tumor suppressor 1 (LATS1) and their common WW domain partners Yes-associated protein (YAP) and the kidney and brain protein KIBRA, we demonstrated by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy that selectivity is achieved via simultaneous interactions as well as mutually exclusive binding. AMOTL1, a primarily disordered protein with three PPxY motifs, simultaneously interacts with YAP and KIBRA via the first and third PPxY motifs respectively. On the contrary, binding of YAP and KIBRA to the two PPxY motif of LATS1 is mutually exclusive, with preferential formation of the YAP-LATS1 complex when all three proteins are present. These discoveries provide exciting new insights into the complex mechanisms that regulate cell growth.