Large Tumor Suppressor Kinase Research Articles

PMEPA1 isoform a drives progression of glioblastoma by promoting protein degradation of the Hippo pathway kinase LATS1

The Hippo signaling pathway controls organ development and is also known, in cancer, to have a tumor suppressing role. Within the Hippo pathway, we here demonstrate, in human gliomas, a functional interaction of a transmembrane protein, prostate transmembrane protein, androgen induced 1 (PMEPA1) with large tumor suppressor kinase 1 (LATS1). We show that PMEPA1 is upregulated in primary human gliomas. The PMEPA1 isoform PMEPA1a was predominantly expressed in glioma specimens and cell lines, and ectopic expression of the protein promoted glioma growth and invasion in vitro and in an orthotopic xenograft model in nude mice. In co-immunoprecipitation experiments, PMEPA1a associated with the Hippo tumor suppressor kinase LATS1. This interaction led to a proteasomal degradation of LATS1 through recruitment of the ubiquitin ligase, neural precursor cell expressed, developmentally downregulated 4 (NEDD4), which led to silencing of Hippo signaling. Alanine substitution in PMEPA1a at PY motifs resulted in failed LATS1 degradation. Targeting of a downstream component in the Hippo signaling pathway, YAP, with shRNA, interfered with the growth promoting activities of PMEPA1a in vitro and in vivo. In conclusion, the presented work shows that PMEPA1a contributes to glioma progression by a dysregulation of the Hippo signaling pathway and thus represents a promising target for the treatment of gliomas.

P314Osteopontin stimulates cell cycle in neonatal cardiomyocytes

Abstract Introduction Macrophages control myocardial regeneration in the neonatal heart and repair in the adult heart. Osteopontin (OPN) is a macrophage-derived protein which is upregulated following tissue injury. While OPN regulates cell adhesion, spreading and migration, its role in myocardial regeneration is unknown. Purpose We aimed to test the hypothesis that OPN stimulates cardiomyocyte proliferation. Methods and results Mouse neonatal cardiomyocytes were treated with a serum-free medium for 24 hours. Immunofluorescent staining showed that these cells expressed the OPN receptor CD44 (Fig. 1A). To assess cell expansion, we used MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colorimetric assay. OPN preserved cardiomyocyte count in a low glucose medium for 24 hours, compared with no OPN treatment (Fig. 1B). Staining for phosphohistone (pH)3, a marker of nucleus division, showed a higher expression of pH3 in cardiomyocytes after OPN treatment (Fig. 1C). Immunoblotting revealed that OPN induced the phosphorylation of cell-cycle activity proteins, known as yes-associated protein (YAP)1, and extracellular signal-regulated kinase (ERK)2, while it decreased the phosphorylation of the YAP1 inhibitor, the large tumor-suppressor kinase (LATS)1/2 (Figs. 1D and 1E). Gene expression analysis revealed that OPN upregulated the transcriptional enhancer factor TEF-1 (TEAD1), which interacts with YAP1 (Fig. 1F), connective tissue growth factor (CTGF) and cyclin-dependent kinase 1 (CDK1), which are YAP downstream targets (Figs. 1G and 1H). Furthermore, OPN significantly upregulated cyclin B1, a regulator of the mitotic (M) phase (Fig. 1I). Moreover, OPN may promote positive feedback by upregulating the expression of CD44, SPP-1 (Figs. 1J and 1K). Finally, administration of OPN to adult mouse improved cardiac remodeling and function after myocardial infarction. Conclusions OPN stimulates cell-cycle activity in neonatal cardiomyocytes through YAP1 activity, independently from the HIPPO-YAP signaling pathway. Because macrophages are a major source of OPN after injury, our findings could explain the essential role of macrophages in neonatal heart regeneration and adult heart repair. Acknowledgement/Funding Israel Science Foundation

Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

Gα13, a heterotrimeric G-protein of the Gα12/13 subfamily, is associated with aggressive phenotypes in various human cancers. However, the mechanisms by which Gα13 promotes cancer progression have not been fully elucidated. Here, we demonstrate that the activation of Gα13 induces epithelial-mesenchymal transition in ovarian cancer (OvCa) cells through down-regulation of large tumor suppressor kinase (LATS) 1, a critical component of the Hippo signaling pathway. A synthetic biology approach using a mutant GPCR and chimeric G-protein revealed that Gα13-regulated phosphorylation of LATS1 at serine 909 within its activation loop induced recruitment of the itchy E3 ubiquitin protein ligase to trigger LATS1 degradation. Our findings uncover novel mechanisms through which Gα13 activation induces dysregulation of the Hippo signaling pathway, which leads to aggressive cancer phenotypes, and thereby identify a potential target for preventing the metastatic spread of OvCa.-Yagi, H., Onoyama, I., Asanoma, K., Hori, E., Yasunaga, M., Kodama, K., Kijima, M., Ohgami, T., Kaneki, E., Okugawa, K., Yahata, H., Kato, K. Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

MiR-16a Regulates Milk Fat Metabolism by Targeting Large Tumor Suppressor Kinase 1 (LATS1) in Bovine Mammary Epithelial Cells.

Milk contains a number of beneficial fatty acids including short and medium chain and unsaturated conjugated and nonconjugated fatty acids. In this study, microRNA sequencing of mammary tissue collected in early-, peak-, mid-, and late-lactation periods was performed to determine the miRNA expression profiles. miR-16a was one of the differentially expressed miRNA and was selected for in-depth functional studies pertaining to fatty acid metabolism. The mimic of miR-16a impaired fat metabolism [triacylglycerol (TAG) and cholesterol] while knock-down of miR-16a promoted fat metabolism in vitro in bovine mammary epithelial cells (BMECs). In addition, the in vitro work with BMECs also revealed that miR-16a had a negative effect on the cellular concentration of cis 9-C18:1, total C18:1, C20:1, and C22:1 and long-chain polyunsaturated fatty acids. Therefore, these data suggesting a negative effect on fatty acid metabolism extend the discovery of the key role of miR-16a in mediating adipocyte differentiation. Through a combination of bioinformatics analysis, target gene 3' UTR luciferase reporter assays, and western blotting, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-16a. Transfection of siRNA-LATS1 into BMECs led to increases in TAG, cholesterol, and cellular fatty acid concentrations, suggesting a positive role of LATS1 in mammary cell fatty acid metabolism. In summary, data suggest that miR-16a regulates biological processes associated with intracellular TAG, cholesterol, and unsaturated fatty acid synthesis through LATS1. These data provide a theoretical and experimental framework for further clarifying the regulation of lipid metabolism in mammary cells of dairy cows.

A kinome-wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion.

The Hippo pathway is an emerging signaling pathway that plays important roles in organ size control, tissue homeostasis, tumorigenesis, metastasis, drug resistance, and immune response. Although many regulators of the Hippo pathway have been reported, the extracellular stimuli and kinase regulators of the Hippo pathway remain largely unknown. To identify novel regulars of the Hippo pathway, in this study we created the first ultra-bright NanoLuc biosensor (BS) to monitor the activity of large tumor suppressor (LATS) kinase 1, a central player of the Hippo pathway. We show that this NanoLuc BS achieves significantly advanced sensitivity and stability both in vitro using purified proteins and in vivo in living cells and mice. Using this BS, we perform the first kinome-wide screen and identify many kinases regulating LATS and its effectors yes-associated protein (YAP) and transcriptional co-activator with PDZ- binding motif (TAZ). We also show for the first time that activation of receptor tyrosine kinase anaplastic lymphoma kinase (ALK) by its extracellular ligand family with sequence similarity (FAM)150 activates Hippo effector YAP/TAZ by increasing their nuclear translocation. Significantly, we show that constitutively active ALK induces tumorigenic phenotypes, such as increased cancer cell proliferation/colony formation via YAP/TAZ and elevated immune evasion via YAP/TAZ-programmed death-ligand 1 in breast and lung cancer cells. In summary, we have developed a new LATS BS for cancer biology and therapeutics research and uncovered a novel ALK-LATS-YAP/TAZ signaling axis that may play important roles in cancer and possibly other biologic processes.-Nouri, K., Azad, T., Lightbody, E., Khanal, P., Nicol, C. J., Yang, X. A kinome-wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion.

Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro†.

Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.

Mono (2-ethylhexyl) phthalate (MEHP) triggers the proliferation of hemangioma-derived endothelial cells via YAP signals

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.

LATS1/2 suppress NFκB and aberrant EMT initiation to permit pancreatic progenitor differentiation.

The Hippo pathway directs cell differentiation during organogenesis, in part by restricting proliferation. How Hippo signaling maintains a proliferation-differentiation balance in developing tissues via distinct molecular targets is only beginning to be understood. Our study makes the unexpected finding that Hippo suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling in pancreatic progenitors to permit cell differentiation and epithelial morphogenesis. We find that pancreas-specific deletion of the large tumor suppressor kinases 1 and 2 (Lats1/2PanKO) from mouse progenitor epithelia results in failure to differentiate key pancreatic lineages: acinar, ductal, and endocrine. We carried out an unbiased transcriptome analysis to query differentiation defects in Lats1/2PanKO. This analysis revealed increased expression of NFκB activators, including the pantetheinase vanin1 (Vnn1). Using in vivo and ex vivo studies, we show that VNN1 activates a detrimental cascade of processes in Lats1/2PanKO epithelium, including (1) NFκB activation and (2) aberrant initiation of epithelial-mesenchymal transition (EMT), which together disrupt normal differentiation. We show that exogenous stimulation of VNN1 or NFκB can trigger this cascade in wild-type (WT) pancreatic progenitors. These findings reveal an unexpected requirement for active suppression of NFκB by LATS1/2 during pancreas development, which restrains a cell-autonomous deleterious transcriptional program and thereby allows epithelial differentiation.

Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

Renal fibrosis is the common pathway of chronic kidney disease progression. The nuclear receptor farnesoid X receptor [FXR, NR1H4 (nuclear receptor subfamily 1 group member 4)], a multifunctional transcription factor, plays a pivotal role in protecting against fibrosis. However, the mechanisms underlying these antifibrotic actions of FXR in kidney disease are largely unknown. Here, we show that agonist GW4064-mediated FXR activation inhibits the activity of the nonreceptor tyrosine kinase Src (proto-oncogene tyrosine-protein kinase), which is critical for regulation of yes-associated protein (YAP) phosphorylation and nuclear localization in renal fibrosis. Activation of FXR suppressed renal fibrosis and Tyr416-Src phosphorylation in TGF-β-treated human renal proximal tubule epithelial (HK2) cells. Moreover, GW4064 treatment in HK2 cells increased Ser127 phosphorylation, cytosolic accumulation of YAP, and interaction of the hippo core kinases (Ste20-like kinase 1, large tumor suppressor kinase 1, and salvador homolog 1). Inhibition of Src using PP2 (Src kinase inhibitor) prevented renal fibrosis and increased Ser127 phosphorylation and cytosolic accumulation of YAP. The expression of fibrosis markers, inflammatory genes, and YAP target genes was increased in the kidneys of FXR knockout mice compared with those of wild-type mice. In addition, GW4064 or WAY-362450 (turofexorate isopropyl) treatment protected against unilateral ureteral obstruction-induced renal fibrosis. Collectively, our data support the novel conclusion that Src-mediated crosstalk between FXR and YAP protects against renal fibrosis, making this pathway a possible therapeutic target for chronic kidney disease.-Kim, D.-H., Choi, H.-I., Park, J. S., Kim, C. S., Bae, E. H., Ma, S. K., Kim, S. W. Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

Hippo signaling pathway regulation of innate immune response

The Hippo signaling pathway plays crucial role in cell proliferation, differentiation, and apoptosis. Furthermore, it acts as a sensor that responds to a variety of environmental conditions, such as glucose starvation, cell density, pathogen infection, and heat shock. Recent studies have revealed that the Hippo signaling pathway is involved in innate immunity. More importantly, this pathway is characterized by the mutual independence of core proteins to regulate innate immunity. In this pathway, the regulation of innate immune response by Yes-associated protein (YAP) is often independent of the large tumor suppressor kinases or mammalian STE20-like kinases. There are further differences in YAP mediation in regulating the innate immune response against pathogens in different tissues and species. In this review, we summarize the evolution and regulation of the Hippo signaling pathway and its role in antiviral and antibacterial defense mechanisms.

MicroRNA-744 promotes carcinogenesis in osteosarcoma through targeting LATS2.

Osteosarcoma (OS) mortality rate is increasing. Various microRNAs (miRNAs) have been investigated in the pathological process of OS except for miR-744. Hence, this research was designed to explore miR-744 function in OS. RT-qPCR and western blot analysis were used to quantify miR-744 and large tumor suppressor kinase 2 (LATS2) expression levels. The function of miR-744 was investigated using MTT and Transwell assays. Target gene of miR-744 was verified by dual-luciferase reporter assay. miR-744 expression was increased in OS, which was associated with worse clinical features and prognosis of OS patients. Importantly, miR-744 promoted cell viability and metastasis in OS. Furthermore, miR-744 induced Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT) in OS. In addition, miR-744 directly targeted LATS2 and blocked its expression in OS. Moreover, upregulation of LATS2 weakened the promotion of cell viability and metastasis induced by miR-744 in OS. In conclusion, miR-744 accelerated OS progression through restraining LATS2 and activating Wnt/β-catenin pathway and EMT.

Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance Induced by Membranous/Cytoplasmic/Nuclear Translocation of Epidermal Growth Factor Receptor

IntroductionThe molecular mechanism underlying the induction of resistance to tyrosine kinase inhibitors (TKIs) via the membranous/cytoplasmic/nuclear translocation of EGFR has not yet been reported. MethodsWe performed immunohistochemistry to detect the distribution of EGFR in lung adenocarcinoma specimens after TKI treatment and analyzed the relationship between different EGFR locations and patient survival duration. Mass spectrometry analysis and immunoprecipitation were performed to show the interaction of cytosolic EGFR with YY1 associated protein 1 (YAP) and salt inducible kinase 2 (SIK2). Dual-luciferase assays, immunoblotting, real-time polymerase chain reaction, and functional experiments were used to elucidate the role of EGFR cytoplasmic/nuclear translocation in Hippo pathway dysregulation. ResultsPatients with advanced lung adenocarcinoma with membranous mutant EGFR (19del or 21 L858R) showed significantly longer progression-free survival than those with cytoplasmic mutant EGFR after gefitinib treatment. The concentration that inhibits 50% in PC-9 with cytoplasmic EGFR was higher than that in hunman non-small cell lung cancer 827 with membranous EGFR. During first-generation TKI resistance induction, membrane EGFR translocated to the cytoplasm/nucleus, accompanied by the Hippo pathway inhibition. Cytoplasmic EGFR and SIK2 interaction inhibited large tumor suppressor kinase 1 (LATS1) and macrophage stimulating 1 (MST1) interaction, promoting YAP nuclear translocation. However, cells with osimertinib-induced resistance also showed EGFR translocation and lower phospho-EGF receptor but did not show Hippo pathway inhibition. Moreover, osimertinib and erlotinib could restore sensitivity to each other in resistant cells. ConclusionsPlasma/nuclear translocation of EGFR and inhibition of the Hippo pathway are some of the important mechanisms underlying the resistance induced by first-generation TKIs. Membrane/plasma translocation of EGFR induced by osimertinib may be another resistance phenomenon besides MNNG HOS transforming gene (c-MET) amplification, C797S mutation, and ERK pathway inhibition.

Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

RETRACTED ARTICLE: Large tumor suppressor kinase 2 overexpression attenuates 5-FU-resistance in colorectal cancer via activating the JNK-MIEF1-mitochondrial division pathway

Background5-Fluorouracil (5-FU) is a standard treatment for colorectal cancer, but most patients develop 5-FU resistance. Here, we conducted experiments to identify an effective approach to augment 5-FU-based treatment in colorectal cancer in vitro.MethodsSW480 cells were in the present study and treated with 5-FU. Besides, LATS2 adenovirus vectors were infected into SW480 cells. Western blotting, immunofluorescence and ELISA were used to evaluate cell death and mitochondrial function. Pathway blocker was used to verify the role of MAPK-JNK pathway in SW480 cell death.ResultsAn obvious drop in large tumor suppressor kinase 2 (LATS2) expression was observed in SW480 cells after treatment with 5-FU. In addition, upregulation of LATS2 expression through infection with LATS2 adenovirus further increased the reduction of SW480 cell viability induced by 5-FU. Functional exploration showed that 5-FU treatment suppressed mitochondrial membrane potential, enhanced cyt-c release into the nucleus, induced an oxidative injury environment by promoting ROS production, and eventually upregulated Bax-related mitochondrial apoptosis. Besides, LATS2 overexpression in combination with 5-FU treatment further perturbed mitochondrial homeostasis, and this effect was achieved by elevating mitochondrial division. Mechanistically, LATS2 overexpression and 5-FU co-treatment amplified mitochondrial division by upregulating MIEF1 expression in a manner dependent on MAPK-JNK axis. Knockdown of MIEF1 using an siRNA-mediated loss of function assay and/or inhibition of the MAPK-JNK pathway using the specific inhibitor SP600125 abolished LATS2/5-FU-mediated deleterious effects on mitochondrial performance and SW480 cell viability.ConclusionsIn light of the above findings, LATS2 downregulation could be a potential mechanism of low response to 5-FU treatment. Overexpression of LATS2 to further disrupt mitochondrial function via the JNK-MIEF1 signalling pathway might be a method to optimize 5-FU-based chemotherapy.

MiR-135b is a novel oncogenic factor in cutaneous melanoma by targeting LATS2.

Melanoma develops from pigment-producing melanocytes in the epidermis, and is the most common type of skin cancer. Because of the lack of effective therapies, the median survival of patients developing metastatic melanoma is less the 1 year. In this paper, we studied the oncogenic role of miR-135b in melanoma cells. We compared the miR-135b levels in 20 melanoma tissues in reference to their corresponding nontumor regions. Next, we studied the impact of miR-135b or its inhibitor on cell proliferation, migration, or apoptosis in either primary melanocytes or the melanoma cell line, respectively. Finally, we validated large tumor suppressor kinase 2 (LATS2) as the downstream target of miR-135b in a luciferase reporter assay, western blotting analysis, and knockdown study in primary melanocytes. MiR-135b expression was significantly upregulated in melanoma tissue. Overexpressing miR-135b in primary melanocytes promoted cell proliferation and migration. In contrast, inhibition of miR-135b expression suppressed the growth and metastasis of A-375 cells and enhanced cell apoptosis. LATS2 was confirmed as the target of miR-135b. Knockdown of LATS2 in melanocytes also promoted cell growth, but not cell invasion potential. Our findings showed miR-135b as a novel oncogene in melanoma tumorigenesis. The oncogenic mechanism may involve the downregulation of LATS2.

LATS2 inhibits cell proliferation and metastasis through the Hippo signaling pathway in glioma.

As a core kinase in the Hippo pathway, large tumor suppressor kinase 2 (LATS2) regulates cell proliferation, migration and invasion through numerous signaling pathways. However, its functions on cell proliferation, migration and invasion in glioma have yet to be elucidated. The present study revealed that LATS2 was downregulated in glioma tissues and cells, as determined by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. In addition, Cell Counting Kit-8, scratch wound healing and Transwell assays revealed that overexpression of LATS2 in U-372 MG cells inhibited cell proliferation, migration and invasion. Furthermore, western blot analysis indicated that the expression levels of phosphorylated (p)-yes-associated protein and p-tafazzin were increased in cells with LATS2 overexpression. These results indicated that LATS2 is a potential tumor suppressor, and downregulation of LATS2 in glioma may contribute to cancer progression.

Citron kinase interacts with LATS2 and inhibits its activity by occluding its hydrophobic phosphorylation motif.

The inhibitory effect of large tumor suppressor kinase (LATS1/2) on the activity of the oncoprotein yes-associated protein (YAP) is crucial to maintain tissue homeostasis. Proteomic studies have identified several new regulators of this process. Recently, citron kinase (CIT) was listed as a potential binding candidate of Hippo-related components, suggesting a new connection between CIT and the Hippo pathway. Aside from CIT’s role in cytokinesis, the molecular crosstalk between CIT and the Hippo pathway is largely unknown. Here, we demonstrate a role for CIT as a scaffold protein linking LATS2 and YAP. More importantly, CIT interacts with LATS2 to directly suppress LATS2 phosphorylation at the hydrophobic motif—targeted by MST1, leading to LATS2 inactivation and YAP activation. By studying their genetic interactions, we found that Sticky, the CIT homolog in Drosophila melanogaster, functions with Warts to control Drosophila eye development. Together, our study confirms citron kinase as a novel regulator of the Hippo pathway.

Expressions of large tumor suppressor kinase 1 and large tumor suppressor kinase 2 in gastric cancer and their significances

Objective To investigate the expressions of large tumor suppressor kinase 1 (LATS1) and large tumor suppressor kinase 2 (LATS2) proteins in gastric cancer tissues, and to explore the correlation between expressions of LATS1 and LATS2 proteins and the occurrence and development of gastric cancer. Methods A total of 93 gastric cancer paraffin tissues and the corresponding adjacent gastric normal mucosa in the Department of Pathology in Baotou Cancer Hospital from September 2008 to June 2010 were collected. The immunohistochemistry was used to detect the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues. The differences of the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues were compared by using χ2 test. The relationship between the expressions of LATS1 and LATS2 proteins and the clinicopathological features was also analyzed. Results In gastric cancer tissues, LATS1 was negatively expressed in 54 cases (58.1%), weakly positive expressed in 15 cases (16.1%), moderately positive expressed in 16 cases (17.2%), and strongly positive expressed in 8 cases (8.6%); in adjacent normal tissues, LATS1 was negatively expressed in 17 cases (18.3%), weakly positive expressed in 16 cases (17.2%), moderately positive expressed in 31 cases (33.3%), and strongly positive expressed in 29 cases (31.2%). The positive expression rate of LATS1 in gastric cancer tissues was lower than that in adjacent normal tissues, and the difference was statistically significant (χ2= 37.460, P 0.05). Conclusion LATS1 and LATS2 proteins may be involved in the occurrence of gastric cancer and have the inhibiting effect on the occurrence and development of gastric cancer. Key words: Gastric neoplasms; Large tumor suppressor kinase 1; Large tumor suppressor kinase 2

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

LATS2 Inhibits Malignant Behaviors of Glioma Cells via Inactivating YAP.

We previously reported that LATS2, the upstream serine/threonine kinase of Yes-associated protein (YAP), is downregulated in gliomas and exhibits negative correlation with the prognosis of glioma patients. In this work, we aimed to explore the role and mechanism of large tumor suppressor kinase (LATS2) in the progression of malignant gliomas. We found that over-expression of LATS2 inhibited glioma cell proliferation and migration/invasion, while LATS2 downregulation promoted them. Mechanistically, LATS2 promoted the phosphorylation of YAP without affecting YAP mRNA expression. Inconsistent with some previous reports, both YAPWT and YAP5SA did not affect the level of LATS2, suggesting that LATS2 did not form a feedback loop with YAP in gliomas. Furthermore, mammalian Ste20-like kinases (MST1) over-expression did not affect the phosphorylation of YAP, suggesting that MST1 may not be the essential upstream serine/threonine kinase of Hippo/YAP pathway in gliomas. Together, LATS2 inhibits glioma cell proliferation and migration/invasion by inactivating YAP.