Large Tumor Suppressor Kinase Research Articles

MiR-497 Regulates LATS1 through the PPARG Pathway to Participate in Fatty Acid Synthesis in Bovine Mammary Epithelial Cells.

Nutrient metabolism is required to maintain energy balance in animal organisms, and fatty acids play an irreplaceable role in fat metabolism. In this study, microRNA sequencing was performed on mammary gland tissues collected from cows during early, peak, and late lactation to determine miRNA expression profiles. Differentially expressed miRNA (miR-497) was selected for functional studies of fatty acid substitution. Simulants of miR-497 impaired fat metabolism [triacylglycerol (TAG) and cholesterol], whereas knockdown of miR-497 promoted fat metabolism in bovine mammary epithelial cells (BMECs) in vitro. In addition, in vitro experiments on BMECs showed that miR-497 could down-regulate C16:1, C17:1, C18:1, and C20:1 as well as long-chain polyunsaturated fats. Thus, these data expand the discovery of a critical role for miR-497 in mediating adipocyte differentiation. Through bioinformatics analysis and further validation, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-497. siRNA-LATS1 increased concentrations of fatty acids, TAG, and cholesterol in cells, indicating an active role of LATS1 in milk fat metabolism. In summary, miR-497/LATS1 can regulate the biological processes associated with TAG, cholesterol, and unsaturated fatty acid synthesis in cells, providing an experimental basis for further elucidating the mechanistic regulation of lipid metabolism in BMECs.

The E3 ubiquitin ligase SCF (FBXW10)-mediated LATS2 degradation regulates angiogenesis and liver metastasis in colorectal cancer.

F-box and WD repeat domain containing 10 (FBXW10) is a member of the FBXW subgroup that contains the WD40 domain. FBXW10 has been rarely reported in colorectal cancer (CRC) and its mechanism is unclear. To investigate the role of FBXW10 in CRC, we conducted in vitro and in vivo experiments. Through the database and our clinical samples, we found that FBXW10 expression was up-regulated in CRC, and it was positively correlated with CD31 expression. CRC patients with high FBXW10 expression levels had a poor prognosis. Overexpression of FBXW10 up-regulated cell proliferation, migration and vascular formation, while knockdown of FBXW10 had the opposite effects. Studies on the mechanism of FBXW10 in CRC showed that FBXW10 could ubiquitinate large tumor suppressor kinase 2 (LATS2) and promote its degradation with the Fbox region of FBXW10 played an essential role in this process. In vivo studies demonstrated that knockout of FBXW10 inhibited tumor proliferation and reduced liver metastasis. In conclusion, our study proved that FBXW10 was significantly overexpressed in CRC and was involved in the pathogenesis of CRC by affecting angiogenesis and liver metastasis. Mechanistically, FBXW10 degraded LATS2 through ubiquitination. Therefore, FBXW10-LATS2 can be used as a therapeutic target for CRC in subsequent studies.

Regulation of Photosensitivity by the Hippo Pathway in Lupus Skin.

Photosensitivity is one of the most common manifestations of systemic lupus erythematosus (SLE), yet its pathogenesis is not well understood. The normal-appearing epidermis of patients with SLE exhibits increased ultraviolet B (UVB)-driven cell death that persists in cell culture. Here, we investigated the role of epigenetic modification and Hippo signaling in enhanced UVB-induced apoptosis seen in SLE keratinocytes. We analyzed DNA methylation in cultured keratinocytes from SLE patients compared to keratinocytes from healthy controls (n=6/group). Protein expression was validated in cultured keratinocytes using immunoblotting and immunofluorescence. An immortalized keratinocyte line overexpressing WWC1 was generated via lentiviral vector. WWC1-driven changes were inhibited using a large tumor suppressor kinase 1/2 (LATS1/2) inhibitor (TRULI) and small interfering RNA (siRNA). The interaction between the Yes-associated protein (YAP) and the transcriptional enhancer associate domain (TEAD) was inhibited by overexpression of an N/TERT cell line expressing a tetracycline-inducible green fluorescent protein-tagged protein that inhibits YAP-TEAD binding (TEADi). Apoptosis was assessed using cleaved caspase 3/7 and TUNEL staining. Hippo signaling was the top differentially methylated pathway in SLE versus control keratinocytes. SLE keratinocytes (n=6) showed significant hypomethylation (Δβ = -0.153) and thus overexpression of the Hippo regulator WWC1 (P = 0.002). WWC1 overexpression increased LATS1/2 kinase activation, leading to YAP cytoplasmic retention and altered proapoptotic transcription in SLE keratinocytes. Accordingly, UVB-mediated apoptosis in keratinocytes could be enhanced by WWC1 overexpression or YAP-TEAD inhibition, mimicking SLE keratinocytes. Importantly, inhibition of LATS1/2 with either the chemical inhibitor TRULI or siRNA effectively eliminated enhanced UVB-apoptosis in SLE keratinocytes. Our work unravels a novel driver of photosensitivity in SLE: overactive Hippo signaling in SLE keratinocytes restricts YAP transcriptional activity, leading to shifts that promote UVB apoptosis.

LATS-regulated nuclear-cytoplasmic translocation of SREBP2 inhibits hepatocellular carcinoma cell migration and invasion via epithelial-mesenchymal transition.

Abnormal cholesterol synthesis plays a crucial role in the development of hepatocellular carcinoma (HCC). Sterol regulatory element-binding protein 2 (SREBP2) is involved in cholesterol synthesis by translocating to the nucleus where it stimulates the transcription of genes encoding enzymes involved in the cholesterol synthesis pathway. However, the function and regulatory mechanism of SREBP2 in HCC remain unclear. In this study, we aimed to gain a better understanding of the effects of SREBP2 and its functional mechanism in HCC. In 20 HCC patients, we demonstrated that SREBP2 was highly expressed in HCC specimens, relative to their peritumoral tissue, and that higher expression correlated positively with a poor prognosis in these patients. Moreover, higher SREBP2 levels in the nucleus enhanced the occurrence of microvascular invasion, whereas inhibition of SREBP2 nuclear translocation by fatostatin markedly suppressed the migration and invasion of HCC cells via the epithelial-mesenchymal transition (EMT) process. The effects of SREBP2 were subject to functional activity of large tumor suppressor kinase (LATS), whereas inhibition of LATS promoted nuclear translocation of SREBP2, as observed in hepatoma cells and a subset of subcutaneous tumor samples from nude mice. In conclusion, SREBP2 enhances the invasion and metastasis of HCC cells by promoting EMT, which can be strengthened by the repression of LATS. Therefore, SREBP2 may serve as a novel therapeutic target for HCC.

Inhibition of EZH2 exerts antitumorigenic effects in renal cell carcinoma via LATS1.

The most common type of kidney cancer in adults is renal cell carcinoma (RCC), which accounts for approximately 90% of cases. RCC is a variant disease with numerous subtypes; the most common subtype is clear cell RCC (ccRCC, 75%), followed by papillary RCC (pRCC, 10%) and chromophobe RCC (chRCC, 5%). To identify a genetic target for all subtypes, we analyzed The Cancer Genome Atlas (TCGA) databases of ccRCC, pRCC, and chromophobe RCC. Enhancer of zeste homolog 2 (EZH2), which encodes a methyltransferase, was observed to be significantly upregulated in tumors. The EZH2 inhibitor tazemetostat induced anticancer effects in RCC cells. TCGA analysis revealed that large tumor suppressor kinase 1 (LATS1), a key tumor suppressor of the Hippo pathway, was significantly downregulated in tumors; the expression of LATS1 was increased by tazemetostat. Through additional experiments, we confirmed that LATS1 plays a crucial role in EZH2 inhibition and has a negative association with EZH2. Therefore, we suggest that epigenetic control could be a novel therapeutic strategy for three subtypes of RCC.

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

Nuclear exclusion of the cotranscription factor YAP, which is a consequence of activation of the Hippo signaling pathway, leads to FSGS and podocyte apoptosis. Ajuba proteins play an important role in the glomerular filtration barrier by keeping the Hippo pathway inactive. In nephrocytes from Drosophila melanogaster , a well-established model system for podocyte research, Ajuba proteins ensure slit diaphragm (SD) formation and function. Hippo pathway activation leads to mislocalization of Ajuba proteins, decreased SD formation, rearrangement of the actin cytoskeleton, and increased SD permeability. Targeting the kinases of the Hippo pathway with specific inhibitors in the glomerulus could, therefore, be a promising strategy for therapy of FSGS. The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS2 regarding expression, localization, interaction, and effects on the functionality of the SD. In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.

Effect of phosphorylation on the assembly of Hippo signaling regulatory complexes.

The Hippo pathway is an evolutionarily conserved signaling network that plays a crucial role in maintaining tissue homeostasis by regulating cell growth/proliferation, differentiation, and apoptosis in response to environmental stimuli. The Hippo pathway integrates a variety of cellular cues including cell contact/density, growth factors, oxidative stress, hormones, and metabolic conditions, to regulate the function of the transcriptional co-activator protein YAP (yes-associated protein) in humans, and its orthologue Yki (yorkie) in Drosophila. Many upstream signals regulate YAP activity by influencing YAP phosphorylation at S127 (S168 in Yki). Although it is understood that phosphorylation of YAP at S127 induces YAP-14-3-3 binding and subsequent cytoplasmic retention, little is known about how phosphorylation at S127 impacts YAP local structure or the assembly of Hippo signaling regulatory complexes. Here we use traditional biophysical techniques such as solution state nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography multi-angle light scattering, to determine the structure, map the binding interface, and characterize the binding thermodynamics of pS127 YAP and Hippo pathway binding partner LATS1 (large tumor suppressor kinase 1).

Changes in Adipose Tissue Gene Expression of the Core Components of the Hippo Signaling Pathway in Young Adults with Uncomplicated Overweight or Obesity Following Weight Loss

BackgroundAppropriate adipogenesis leads to the “healthy” expansion of adipose tissue and is a crucial component in maintaining metabolic homeostasis. The Hippo signaling network may balance adipocyte proliferation/differentiation regulating adipogenic footpath. ObjectivesOur study aimed to assess subcutaneous adipose tissue (SAT) expression of genes involved in Hippo signaling network in subjects with marked overweight or obesity after dietary intervention (DI) in relation to obesity and insulin sensitivity. MethodsForty overweight or obese subjects (O/O) [mean ± SD age 33 ± 7 y, 45% men, BMI (in kg/m2) 32.9 ± 3.1] completed DI [low-calorie diet (20 kcal/kg of proper body weight) for 12 wks]. The control group comprising 20 normal-weight subjects (mean ± SD age: 24 ± 2 y, 40% men, BMI: 22.4 ± 2.3 ) was examined at baseline only. Hyperinsulinemic-euglycemic clamp and SAT biopsy with gene expression analysis were performed. Student’s t-test for unpaired and paired samples and Pearson correlation analysis were applied. This is an exploratory analysis of the DI program. ResultsSAT mRNA expression of mammalian sterile 20-like kinase 2 (MST2) encoded by serine/threonine kinase 3 gene (STK3)-->, large tumor suppressor kinase 2 (LATS2), and salvador family WW domain containing protein 1 (SAV1), the upstream members of the Hippo pathway, were decreased (21%, 40%, and 36%, respectively) in O/O in comparison with weight subjects individuals before DI (all P < 0.05). At baseline, positive correlations between SAT SAV1, LATS2 expression and adiponectin (ADIPOQ) (r = 0.50, P < 0.001; r = 0.53, P = 0.004, respectively) and solute carrier family 2 member 4 (SLC2A4) (r = 0.35, P = 0.007; r = 0.28, P = 0.03, respectively) expression were observed in the entire study group. Body weight of the O/O group decreased during DI (11.2 ± 3.8 kg, P < 0.001), and there was an increase in insulin sensitivity (by 27%) and SAT expression of STK3, LATS2 (both by 19%), and SAV1 (by 26%) (all P < 0.05). After DI, SAT SLC2A4 expression was correlated with STK3 (r = 0.47, P = 0.003), LATS2 (r = 0.56, P < 0.001), and yes-associated protein (r = 0.50, P = 0.001) expression. ConclusionsObesity is associated with altered mRNA expression of upstream effectors of the Hippo pathway in SAT in young adults. DI may improve adipogenic capacity. J Nutr 20XX;xx:xx–xx.

Prolactin promotes crop epithelial proliferation of domestic pigeons (Columba livia) through the Hippo signaling pathway.

The purpose of this study was to investigate whether prolactin (PRL) regulates the proliferation of pigeon crop epithelium through the Hippo signaling pathway during the breeding cycle. Twenty-four pairs of adult pigeons were allotted to four groups by different breeding stages, and their crops and serum were sampled. Eighteen pairs of young pigeons were selected and divided into three groups for the injection experiments. The results showed that the serum PRL content and crop epithelial thickness of pigeons increased significantly at day 17 of incubation (I17) and day 1 of chick-rearing (R1). In males, the mRNA levels of yes-associated transcriptional regulator (YAP) and snail family transcriptional repressor 2 (SNAI2) were peaked at I17, and the gene levels of large tumor suppressor kinase 1 (LATS1), serine/threonine kinase 3 (STK3), TEA domain transcription factor 3 (TEAD3), connective tissue growth factor (CTGF), MYC proto-oncogene (c-Myc) and SRY-box transcription factor 2 (SOX2) reached the maximum value at R1. In females, the gene expression of YAP, STK3, TEAD3, and SOX2 reached the greatest level at I17, the expression profile of SAV1, CTGF, and c-Myc were maximized at R1. In males, the protein levels of LATS1 and YAP were maximized at R1 and the CTGF expression was upregulated at I17. In females, LATS1, YAP, and CTGF reached a maximum value at I17, and the expression level of phosphorylated YAP was minimized at I17 in males and females. Subcutaneous injection of prolactin (injected for 6 d, 10 μg per kg body weight every day) on the left crop of pigeons can promote the proliferation of crop epithelium by increasing the CTGF level and reducing the phosphorylation level of YAP. YAP-TEAD inhibitor verteporfin (injection for 6 d, 2.5 mg per kg body weight every day) can inhibit the proliferation of crop epithelium induced by prolactin by inhibiting YAP and CTGF expression. In conclusion, PRL can participate in crop cell proliferation of pigeons by promoting the expression of YAP and CTGF in Hippo pathway.

Clinicopathological Significance of DUB3 Expression in Non-small Cell Lung Cancer and Relationship Between DUB3 Expression and LATS1 Expression.

Deubiquitinating enzyme 3 (DUB3) is a member of the ubiquitin-specific proteases family involved in regulating cell proliferation, invasion, and apoptosis. However, the biological role and clinicopathological significance of DUB3 expression have not been elucidated in non-small cell lung cancer (NSCLC). We evaluated the expression of DUB3 by immunohistochemistry using tissue microarrays and assessed the clinicopathologic significance of DUB3 expression levels in 187 patients with NSCLC, including its two major subtypes (93 cases of adenocarcinoma and 72 cases of squamous cell carcinoma). In adenocarcinoma, we observed that DUB3 expression had an effect on tumor size (p=0.030), vessel invasion (p=0.038), T stage (p=0.014), and tumor recurrence (p=0.002). Kaplan-Meier curves with log-rank test showed that high DUB3 expression was correlated with significantly more favorable clinical outcomes compared to those of the low expression group in adenocarcinoma (p=0.013). Multivariate analysis of disease-free survival also demonstrated that DUB3 expression is an independent prognostic factor in lung adenocarcinoma (p=0.017). Additionally, we identified the correlation between DUB3 and the expression of large tumor suppressor kinase 1 expression, a core protein of the Hippo pathway. DUB3 could function as a tumor suppressor by regulating the Hippo pathway in lung adenocarcinoma and can be considered a powerful predictive factor and therapeutic target.

MICRORNA-31 AS A POTENTIAL THERAPEUTIC BIOMARKER FORORAL SQUAMOUS CELL CARCINOMA: CURRENT EVIDENCE AND FUTURE PROSPECTS.

Dear Editor,&#x0D; Cancer is a foremost death cause in each country and a substantial hindrance to improving life expectancy, accounting for more than 10 million fatalities in 2020, or around one in every six fatalities [1]. Oral cancer is among the leading causes of cancer-related mortality in developing countries. Oral squamous cell carcinoma (OSCC) is a widespread type of malignancy associated with tobacco usage and human papilloma virus infection. The majority of people are diagnosed with cancer at an advanced stage, the 5-year survival rate for OSCC patients is only around 50%. Despite biological and technological advances, in recent decades, the prognosis for OSCC has not improved, and its occurrence has increased. Identification of biomarkers that can be utilized to establish an early diagnosis or predict tumor growth is enabled by determining which molecular pathways are disrupted [2].&#x0D; MicroRNAs, which can serve as both oncogenes and tumor-inhibiting factors, are misaligned in a wide range of cancers [3]. These are short, noncoding RNAs that affect protein expression following transcription at the post-transcriptional level. MicroRNAs play a vital role in proliferation, differentiation, apoptosis, immune response, and angiogenesis, among other things in the pathogenesis of cancer. Multiple researchers have documented that specific microRNAs are frequently down regulated in cancers, implying that they operate as tumor suppressors by targeting many oncogenes. According to research conducted by O’Brien et al, altering levels of microRNAs expression can improve chemotherapeutic efficacy [4]. In recent decades, several researches have linked metastasis, survival, and recurrence of OSCC patients to miRNA expression [5].&#x0D; MicroRNA-31, a gene on chromosome 9p21.3, was one of the first mammalian microRNAs discovered. Several studies have been conducted to evaluate the functions of miR-31 in carcinogenesis and the progression of OSCC. A prior clinical investigation conducted by Liu et al. [6] revealed that miR-31 levels in plasma were considerably increased in patients with OSCC when compared to age and gender-matched control participants. Several studies show that miR-31 performs a role in cancer etiology and aggressiveness via altering target genes. According to reports, miR-31 regulates a number of genes, including fibronectin type III domain containing 5, special AT-rich sequence-binding protein-2, large tumor suppressor kinase 2 (LATS2), tensin 1, AT-rich interaction domain 1A, and hypoxia-inducible factor-1 [7]. The concentration of miR-31 expression in serum, urine, saliva and organ tissue can be utilized as a diagnostic and predictive biomarker for a variety of malignancies [8].&#x0D; A recent investigation by Chou et al. [9] found that miR-31 levels were significantly raised in OSCC cells and tumor tissues, and that the miR-31 gene locus was necessary to initiate oncogenesis in OSCC. Upregulation of miR-31 contributes to the formation of OSCC by interacting with certain genes and signaling pathways that impact cancer cell cycle, epithelial-mesenchymal transition and cell proliferation. miR-31 forms a complex network with direct target genes (such as RHOA, FIH, ACOX1, VEGF, SIRT3, LATS2, KANK1, and NUMB) and initiates several signaling pathways including the ERK-MMP9 cascade, the Hippo pathway, Wnt signaling, and the MCT1/MCT4 regulatory cascade [10]. MiR-31 interacts with a number of proteins and pathways that are crucial in OSCC and it is overexpressed in plasma, saliva, and OSCC tumor tissue. However, more research is needed to fully comprehend the molecular mechanisms by which miR-31 drives OSCC malignancy, paving the way for the development of miR-31 based diagnostic, prognostic, and predictive biomarkers for OSCC.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

LncRNA surfactant associated 1 activates large tumor suppressor kinase 1/Yes-associated protein pathway via modulating hypoxic exosome-delivered miR-4766-5p to inhibit lung adenocarcinoma metastasis.

LncRNA surfactant associated 1 (SFTA1P) exhibits low expression in non-small cell lung cancer (NSCLC) tissues as compared with that in adjacent tissues, and may play a suppressing role in NSCLC. However, the effect and mechanism of SFTA1P on the metastasis of lung adenocarcinoma (LUAD) remain undefined, which are thus investigated in this research. Herein, potential impacts of SFTA1P on LUAD were determined through the Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA). After knockdown/overexpression of SFTA1P, the metastatic ability of LUAD cells was evaluated by molecular biology experiments (cell counting kit-8 assay, scratch test, Transwell assay and Western blot). The effect of SFTA1P on Yes-associated protein (YAP) nuclear translocation was assessed by Western blot. Hypoxia-induced exosomes were extracted for LUAD metastasis analysis. The targeting relationship of SFTA1P/miR-4766-5p/large tumor suppressor kinase 1 (LATS1) was verified by dual-luciferase reporter assay and molecular biology experiments. Xenograft and lung metastasis models were constructed for in vivo validation. SFTA1P was lowly expressed in LUAD, which was associated with the poor prognosis of patients with LUAD. Up-regulated SFTA1P prevented the metastasis of LUAD cells and the nuclear translocation of YAP. Hypoxia-induced exosomes stimulated LUAD cell metastasis, but inhibited the SFTA1P and LATS1/YAP axes. MiR-4766-5p acted as an intermediate "bridge" for SFTA1P to regulate LATS1. SFTA1P repressed xenograft growth and LUAD cell metastasis. To sum up, SFTA1P activates hypoxic exosome-delivered miR-4766-5p through modulating LATS1/YAP pathway, thereby suppressing LUAD cell metastasis, which may serve as a suitable target for the LUAD therapy.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundanceof WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Verteporfin inhibits the dedifferentiation of tubular epithelial cells via TGF-β1/Smad pathway but induces podocyte loss in diabetic nephropathy

AimsThe dedifferentiation of tubular epithelial cells has been identified as an important trigger of renal fibrosis. The Hippo pathway is a crucial regulator of cell proliferation and differentiation. In this study, we determined the role of Hippo proteins in tubular dedifferentiation in diabetic nephropathy (DN). Main methodsIn this study, we measured dedifferentiation markers and Hippo proteins in db/db mice and high glucose treated tubular epithelial cells. Then, verteporfin and knockdown of large tumor suppressor kinase (LATS) 1 and 2 were performed to uncover therapeutic targets for DN. Key findingsHere, we found dedifferentiation and upregulated Hippo proteins in tubular epithelial cells in DN model both in vivo and in vitro. Both verteporfin and LATS knockdown could inhibit the tubular mesenchymal transition, but verteporfin showed broad inhibitory effect on Hippo proteins, especially nuclear YAP, and exacerbated podocyte loss of DN. LATS2 knockdown did not reverse the tubular E-Cadherin loss while it also induced podocyte apoptosis. Overall, intervention of LATS1 inhibited tubular dedifferentiation efficiently without affecting YAP and bringing podocyte apoptosis. Further mechanistic investigations revealed that the TGF-β1/Smad, instead of the YAP-TEAD-CTGF signaling, might be the underlying pathway through which verteporfin and LATS1 engaged in the tubular dedifferentiation. SignificanceIn conclusion, verteporfin is not a suitable treatment for DN owing to evitable podocyte loss and apoptosis. Targeting LATS1 is a better choice worthy of further investigation for DN therapy.

Neratinib as a Potential Therapeutic for Mutant RAS and Osimertinib-Resistant Tumours

Neratinib was developed as an irreversible catalytic inhibitor of ERBB2, which also acts to inhibit ERBB1 and ERBB4. Neratinib is U.S. Food and Drug Administration (FDA)-approved as a neo-adjuvant therapy for use in HER2+ breast cancer. More recently, chemical biology analyses and the authors’ own bench work have demonstrated that neratinib has additional targets, which open up the possibility of using the drug in cell types that either lack ERBB receptor family expression or who rely on survival signalling downstream of growth factor receptors. Neratinib rapidly disrupted mutant RAS nanoclustering, which was followed by mutant rat sarcoma virus proteins translocating via LC3-associated phagocytosis into the cytosol where they were degraded by macroautophagy. Neratinib catalytically inhibited the MAP4K mammalian STE20-like protein kinase 4 and also caused its degradation via macroautophagy. This resulted in ezrin dephosphorylation and the plasma membrane becoming flaccid. Neratinib disrupted the nanoclustering of RAC1, which was associated with dephosphorylation of PAK1 and Merlin, and with increased phosphorylation of the Merlin binding partners large tumour suppressor kinase 1/2, YAP, and TAZ. YAP and TAZ exited the nucleus. Neratinib retained its anti-tumour efficacy against NSCLC cells made resistant to either afatinib or to osimertinib. Collectively, these findings argue that the possibilities for the further development of neratinib as cancer therapeutic in malignancies that do not express or over-express members of the ERBB receptor family are potentially wide-ranging.

The Hippo pathway mediates Semaphorin signaling.

Semaphorins were originally identified as axonal guidance molecules, but they also control processes such as vascular development and tumorigenesis. The downstream signaling cascades of Semaphorins in these biological processes remain unclear. Here, we show that the class 3 Semaphorins (SEMA3s) activate the Hippo pathway to attenuate tissue growth, angiogenesis, and tumorigenesis. SEMA3B restoration in lung cancer cells with SEMA3B loss of heterozygosity suppresses cancer cell growth via activating the core Hippo kinases LATS1/2 (large tumor suppressor kinase 1/2). Furthermore, SEMA3 also acts through LATS1/2 to inhibit angiogenesis. We identified p190RhoGAPs as essential partners of the SEMA3A receptor PlexinA in Hippo regulation. Upon SEMA3 treatment, PlexinA interacts with the pseudo–guanosine triphosphatase (GTPase) domain of p190RhoGAP and simultaneously recruits RND GTPases to activate p190RhoGAP, which then stimulates LATS1/2. Disease-associated etiological factors, such as genetic lesions and oscillatory shear, diminish Hippo pathway regulation by SEMA3. Our study thus discovers a critical role of Hippo signaling in mediating SEMA3 physiological function.

Identification of NUAK1/2 Regulators in the Hippo Signaling Pathway

The Hippo signaling pathway plays a central role in regulating cell proliferation, differentiation, and tissue size. In response to various upstream regulators, the mammalian STE20‐like protein kinase 1/2 (MST1/2) and large tumor suppressor kinase 1/2 (LATS1/2) phosphorylate Yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ) to promote their cytoplasmic retention and degradation. Unphosphorylated YAP/TAZ translocate into the nucleus and interact with several transcription factors to regulate gene expression that drives cell growth and migration. Dysregulation of the Hippo pathway causes tissue overgrowth and tumorigenesis and elevated YAP/TAZ levels or activities are positively correlated with progression of almost all solid cancers. The NUAK family kinase 1/2 (NUAK1/2) are members of the AMP‐activated protein kinase‐related kinase family. Our lab showed that NUAK2 negatively regulates the Hippo pathway by preventing LATS‐mediated YAP/TAZ phosphorylation and cytoplasmic sequestration. Moreover, this work showed that loss of NUAK2 attenuates in vivo mammary tumor growth. However, how NUAK1/2 are regulated is poorly understood.Here, we will undertake screens in cancer cell lines to identify upstream and downstream NUAK1/2 regulators and partners. Since, abrogation of NUAK expression blocks cell growth in a context‐dependent manner, my current efforts are being directed towards generating cell lines expressing inducible NUAK1/2 shRNAs. Independent clones have been selected and validated for their knockdown efficiencies and effects on YAP/TAZ downstream target gene expression. Once validated, the selected lines will be subjected to genome‐wide CRISPR screens. We hypothesize that the screens will identify components that enhance or attenuate the effect of NUAKs and will reveal any differential regulators and partners for NUAK1/2. Discovering novel cancer‐relevant NUAK regulators will enhance our understanding of the tumor promoting roles of NUAKs and provide insights into how to target these kinases for better therapeutic outcomes.

RP1-59D14.5 triggers autophagy and represses tumorigenesis and progression of prostate cancer via activation of the Hippo signaling pathway

Prostate cancer (PCa) is one of the major malignant tumors among men worldwide. Long noncoding RNAs (lncRNAs) have been documented as important modulators in human cancers, including PCa. In our study, we investigated the role and potential mechanism of RP1-59D14.5 in PCa. RP1-59D14.5 expressed at a low level in PCa cells. Gain-of-function assays including colony formation and transwell assays displayed that RP1-59D14.5 overexpression repressed PCa cell proliferation, migration, and invasion. Besides, RP1-59D14.5 up-regulation induced autophagy in PCa cells. Mechanically, luciferase reporter assays and western blot verified that RP1-59D14.5 activated the Hippo pathway in PCa cells. Through RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays, we validated that RP1-59D14.5 functioned as a competing endogenous RNA (ceRNA) to regulate large tumor suppressor kinase 1/2 (LATS1/2) via targeting miR-147a. Moreover, RP1-59D14.5 recruited HUR to promote casein kinase 1 (CK1) expression. Collectively, RP1-59D14.5 promoted yes-associated protein (YAP) degradation to activate the Hippo pathway in PCa progression via targeting the miR-147a/LATS1/2 axis and recruiting HUR to promote the interaction of CK1 and β-transducin repeat-containing protein (βTrCP). These results implied that RP1-59D14.5 acted as a tumor suppressor in PCa, which might be a target for PCa treatment.

Antitumor Activity of lncRNA NBAT-1 via Inhibition of miR-4504 to Target to WWC3 in Oxaliplatin-Resistant Colorectal Carcinoma.

Background Increasing evidence shows that dysfunction of noncoding RNAs is implicated in cancer. Neuroblastoma associated transcript 1 (NBAT-1) has been identified as a tumor suppressive lncRNA that is aberrantly expressed in cancers. However, the function and the underlying mechanisms of the NBAT-1 in colorectal carcinoma (CRC) remain unknown. Methods Gene expression was detected by RT-qPCR. The influence of NBAT-1 on CRC was evaluated by the cell counting kit-8 (CCK-8) assay and an in vivo xenograft mouse model. The possible binding of NBAT-1 to miRNAs was predicted via the miRDB online tool and confirmed by a dual-luciferase reporter assay. Protein expression was detected by western blot. Results NBAT-1 expression was significantly decreased in CRC tissues, especially in patients with oxaliplatin (OXA) resistance. NBAT-1 inhibited OXA-resistant CRC cell proliferation in vitro and tumor growth in vivo. The mechanism study revealed that NBAT-1 functioned as a competing endogenous RNA (ceRNA) of miR-4504. NBAT-1 bound miR-4504 and decreased miR-4504 expression in CRC cells. Furthermore, WW-and-C2-domain-containing protein family member 3 (WWC3) was identified as a target of miR-4504. Downregulation of NBAT-1 promoted miR-4504 expression and reduced the level of WWC3. Inhibition of WWC3 by NBAT-1 depletion inactivated Hippo signalling by inhibiting the phosphorylation of large tumor suppressor kinase 1 (LATS1) and yes-associated protein (YAP). Consistently, knockdown of NBAT-1 suppressed the expression of YAP transcriptional targets. Conclusions These findings demonstrated that lncRNA NBAT-1 suppresses OXA-resistant CRC cell growth via inhibition of miR-4504 to regulate the WWC3/LATS1/YAP axis.