Mammalian Sterile 20-like Kinases Research Articles

Amplification of Hippo Signaling Pathway Genes Is Governed and Implicated in the Serous Subtype-Specific Ovarian Carcino-Genesis.

Among women, ovarian cancer ranks as the fifth most common cause of cancer-related deaths. This study examined the impact of Hippo signaling pathway on ovarian carcinogenesis. Therefore, the signatures related to Hippo signaling pathway were derived from the molecular signatures database (MSigDB) and were used for further analysis. The Z score-based pathway activation scoring method was employed to investigate the expression patterns of these signatures in the mRNA expression profiles of ovarian cancer cohorts. Compared to other subtype tumors, the results of this study show that the Hippo signaling pathway signatures are dysregulated prominently in serous subtype-specific ovarian carcinogenesis. A receiver operating characteristic (ROC) curve-based results of the Hippo gene set, yes-associated protein 1 (YAP1), and mammalian sterile 20-like kinases 1 (MST1) genes can predict the serous subtype tumors by higher specificity and sensitivity with significant areas under the curve values also further reconfirmed these signaling dysregulations. Moreover, these gene sets were studied further for mutation analysis in the profile of high-grade serous ovarian adenocarcinoma in the cBioPortal database. The OncoPrint results reveal that these Hippo signaling pathway genes are amplified highly during the grade three and stage third or fourth of serous type ovarian tumors. In addition, the results of the Dependency Map (DepMap) plot also clearly show that these genes are amplified significantly across the ovarian cancer cell lines. Finally, overall survival (OS) curve plot investigations also revealed that these gene expressions show poor survival patterns linked to highly expressed conditions in serous subtypes of ovarian cancer patients with significant p-values (p < 0.05). Thus, the current finding would help to develop the targeted therapies treatment for serous subtype ovarian carcinogenesis.

Syndecan-2 modulates the YAP pathway in epithelial-to-mesenchymal transition-related migration, invasion, and drug resistance in colorectal cancer

Epithelial-to-mesenchymal transition (EMT) is associated with an invasive phenotype in colorectal cancer (CRC). Here, we examined the roles of YES-associated protein (YAP) and syndecan-2 (SDC2) in EMT-related progression, invasion, metastasis, and drug resistance in CRC. The expression levels of YAP and SDC2 in CRC patient tumor tissue were quantified by PCR and western blotting. EMT-associated characteristics were assessed using Transwell assays and immunohistochemistry. Co-immunoprecipitation, glutathione S-transferase pull-down, and luciferase reporter assays were used to assess interactions between YAP and SDC2. YAP was found to be highly expressed in tumor tissue from 13/16 CRC patients, while SDC2 was highly expressed in the tumor tissue of 12/16 CRC patients. Overexpression of YAP in colon cancer cells led to increased cell viability, invasion, migration, and oxaliplatin resistance demonstrating that YAP plays a role in EMT. In addition, overexpression of YAP led to decreased expression of the large tumor suppressor kinase 1 (LATS1) and mammalian sterile 20-like kinases (MST1/2). Decreased LATS1 expression was associated with increased levels of cell proliferation. Knockdown of YAP by shRNA interference led to decreased cell invasion, migration, and drug resistance in colon cancer cells and reduced tumorigenesis in a mouse xenograft model. Finally, we established that YAP interacted with SDC2, and demonstrated that SDC2 mediated the YAP pathway through the EMT-related factors BMP4, CTGF and FOXM1.

Gut microbiota transplantation from db/db mice induces diabetes-like phenotypes and alterations in Hippo signaling in pseudo germ-free mice.

Type 2 diabetes mellitus (T2DM) is an age-related metabolic disease that is of increasing concern. Gut microbiota might have a critical role in the pathogenesis of T2DM. Additionally, Hippo signaling has been associated strongly with the progression of T2DM and the aging process. We adopted db/db male mice as a T2DM model, and the gut microbiota of db/db and m/m mice were transplanted successfully into pseudo germ-free mice. Furthermore, Hippo signaling, including mammalian sterile 20-like protein kinases 1 (MST1), large tumor suppressors 1 (LATS1), Yes-associated protein (YAP), and phosphorylation of YAP (p-YAP) in peripheral tissues were significantly altered and highly correlated with blood glucose in db/db mice. Interestingly, the host after gut microbiota transplantation from db/db mice showed decreased MST1 and LATS1 levels, and p-YAP/YAP ratio in the heart, liver, and kidney compared to those from m/m mice. Negative correlations between fasting blood glucose and Hippo signaling levels in selected peripheral tissues also were identified. These findings suggest that alterations in Hippo signaling in selected peripheral tissues may contribute to the development of T2DM, and that therapeutic interventions improving Hippo signaling by gut microbiota transplantation might be beneficial for the treatment of T2DM and other age-related metabolic diseases.

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway.

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.

FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2+ breast cancer cells likewise led to increased MST1/2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2+ MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2+ breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2-induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.

Mammalian Sterile20-like Kinases: Signalings and Roles in Central Nervous System.

Mammalian Sterile20-like (MST) kinases are located upstream in the mitogen-activated protein kinase pathway, and play an important role in cell proliferation, differentiation, renewal, polarization and migration. Generally, five MST kinases exist in mammalian signal transduction pathways, including MST1, MST2, MST3, MST4 and YSK1. The central nervous system (CNS) is a sophisticated entity that takes charge of information reception, integration and response. Recently, accumulating evidence proposes that MST kinases are critical in the development of disease in different systems involving the CNS. In this review, we summarized the signal transduction pathways and interacting proteins of MST kinases. The potential biological function of each MST kinase and the commonly reported MST-related diseases in the neural system are also reviewed. Further investigation of MST kinases and their interaction with CNS diseases would provide the medical community with new therapeutic targets for human diseases.

RAS Association Domain Family Member 4 (RASSF4): A New Potent Tumor Suppressor in Multiple Myeloma

Multiple myeloma (MM) is a genetic highly heterogeneous plasma cell malignancy. In MM, the RAS/MAPK pathway is the most frequently mutated pathway, leading to aberrant MEK/ERK and PI3K/Akt signaling and thus stimulating MM cell growth and survival. Increasing evidence indicates that next to their classical oncogenic effects, RAS proteins can also induce growth inhibitory effects and apoptosis. These tumor suppressive effects are mainly mediated through the Ras-Association Domain Family (RASSF), a group of 10 RAS effector proteins (RASSF1-10) and numerous isoforms. Based on the localization of the Ras-association (RA) domain this family is further subdivided into two groups. RASSF1 to 6 harbor a C-terminal RA domain and are termed the classical group, while RASSF7-10 contain a N-terminal RA domain and are referred to as non-classical RASSF proteins. The classical RASSF members also carry a SARAH domain on the C-terminus, allowing the RASSF proteins to bind each other and the mammalian sterile 20-like kinases MST and MST-2. Currently, no data about the biological role of RASSF proteins in the pathogenesis of MM is available.Here, we first investigated the expression of the classical RASSF proteins RASSF1-6 in MM using publically available gene expression profiling data of 2 independent newly diagnosed patient cohorts (namely the Heidelberg-Montpellier and the UAMS-TT2 cohort) and 1 cohort of relapsed patients (Mulligan-cohort). We report that RASSF4 was the only classical RASSF protein that was consistently found downregulated in MM cells compared to normal bone marrow plasma cells, correlating with a bad prognosis in all cohorts. Treatment with epigenetic modulating agents including histone deacetylase inhibitors (HDACi) and DNA methyltransferases inhibitors significantly increased RASSF4 expression both in vitro and in vivo, indicating that RASSF4 downregulation in MM is due to epigenetic silencing. Forced RASSF4 expression induced a strong G2-phase arrest and caspase-3 mediated apoptosis in human MM cell lines and strongly reduced the viability of primary CD138+ MM cells. Moreover, RASSF overexpression significantly reduced in vivotumor growth. In addition, we showed RASSF4-MST1 binding and the activation of the downstream signaling pathways JNK/Jun, p38 and p53. RNA sequencing and gene set enrichment analysis following forced RASSF4 expression furthermore revealed downregulation of genes involved in protein metabolism, the unfolded protein response and translation. RASSF4 overexpression also sensitized MM cells to the proteasome inhibitor bortezomib, the specific MEK1/2 inhibitor trametinib and the ROS inducer Prima-1Met. Consequently, combining trametinib with HDACi, e.g. panobinostat, resulted in very strong synergistic anti-MM effects.In conclusion, we identified RASSF4 as a new potent tumor suppressor in MM that is epigenetically silenced in malignant plasma cells and provide a rationale for testing novel therapeutic strategies enhancing RASSF4 expression (using HDACi or gene therapy) in combination with MEK/ERK inhibitors and bortezomib. DisclosuresHose:Takeda: Other: Travel grant; EngMab: Research Funding; Sanofi: Research Funding.

Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway.

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 3₁₀-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.

Abstract 18817: The Role of Mst1 and Mst2 in Pressure Overload-Induced Hypertrophy and Failure

Mammalian sterile 20-like kinases 1 and 2 (Mst1/2) are highly conserved and are thought to have similar pro-apoptotic and anti-cell growth properties. Previous work has demonstrated that increased expression of Mst1 causes robust cardiomyocyte apoptosis, heart failure and premature death in mice, yet the function of endogenous Mst1/2 has not been investigated. The goal of this study was to determine the role of Mst1 and Mst2 in the development of heart failure using genetically altered Mst isoform-specific mice. Systemic deletion of both Mst isoforms is embryonic lethal; therefore we utilized mice harboring floxed Mst1 alleles on an Mst2-/- background and bred them with α-MHC Cre mice to disrupt both isoforms in cardiomyocytes (Mst DKO). Animals were subjected to transverse aortic constriction for 4 weeks or sham. Echocardiography was performed prior to intervention and immediately before sacrifice. At 8 weeks of age, no significant differences in the cardiac phenotype were observed between control, Mst1-/-, Mst2-/- or Mst DKO mice. In response to pressure overload (PO), control mice had increased heart weight/tibia length (HW/TL), left ventricle weight/tibia length (LVW/TL) and cardiomyocyte cross-sectional area as expected. Surprisingly, this hypertrophic response was significantly attenuated in Mst1-/- and Mst2-/- mice. Interestingly, Mst DKO mice hypertrophied similar to controls. Apoptosis and fibrosis were increased, and LV ejection fraction (LVEF) was decreased after PO in control mice. Mst1-/- and Mst2-/- mice had attenuated apoptosis and fibrosis; however, LVEF was decreased to levels similar to controls. Interestingly, Mst DKO mice also showed attenuated apoptosis and fibrosis but had significantly worse LVEF compared to control mice. Studies are ongoing to address the mechanism responsible for worsened cardiac function observed in Mst DKO mice despite suppression of fibrosis and apoptosis after PO.

Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

BackgroundStriatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases.ResultsTo help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3.ConclusionsStriatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between striatin's calmodulin-binding and WD-domains and recruits the PP2A A/C heterodimer to its coiled-coil/oligomerization domain. Residues outside the previously reported coiled-coil domain of striatin are necessary for its oligomerization. Striatin-associated PP2A is critical for Mst3 dephosphorylation and inactivation. Upon inhibition of PP2A, Mst3 activation appears to involve autophosphorylation of multiple activation loop phosphorylation sites. Mob3 can associate with striatin sequences C-terminal to the Mst3 binding site but also with sequences proximal to striatin-associated PP2A, consistent with a possible role for Mob 3 in the regulation of Mst3 by PP2A.

Roles of mammalian sterile 20-like kinase 2-dependent phosphorylations of Mps one binder 1B in the activation of nuclear Dbf2-related kinases

Mammalian nuclear Dbf2-related (NDR) kinases (LATS1, LATS2, NDR1 and NDR2) play a role in cell proliferation, apoptosis and morphological changes. Mammalian sterile 20-like (MST) kinases and Mps one binder (MOB) proteins are important in the activation of NDR kinases. MOB1 is phosphorylated by MST1 and MST2 and this phosphorylation enhances the ability of MOB1 to activate NDR kinases. The phosphorylated MOB1 can be more effective as a scaffold protein to facilitate the MST-dependent phosphorylation of NDR kinases and/or as a direct activator of NDR kinases. We previously reported that Thr74 of MOB1B is phosphorylated by MST2. Thr12 and Thr35 have also been identified as phosphorylation sites. In this study, we quantified the phosphorylation of Thr74 using the phosphorylated Thr74-specific antibody. Thr74 is indeed phosphorylated by MST2, but the efficiency is low, suggesting that MOB1B can activate NDR kinases without the phosphorylation of Thr74. We also showed that the phosphorylated MOB1B activates NDR1 T444D and LATS2 T1041D, in which threonine residues phosphorylated by MST kinases are replaced with phosphorylation-mimicking aspartic acid, more efficiently than the unphosphorylated MOB1B does. This finding supports that the phosphorylation of MOB1B enhances its ability as a direct activator of NDR kinases.

Threonine 74 of MOB1 is a putative key phosphorylation site by MST2 to form the scaffold to activate nuclear Dbf2-related kinase 1

Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.