Ste20-like Kinase Research Articles

YAP promotes AP-1 expression in tubular epithelial cells in the kidney.

Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.

Genetic variants in Mammalian STE20-like protein kinase 2 were associated with risk of NSCL/P

AimMammalian STE20-like protein kinase 2 (MST2) plays an important role in apoptosis and the development of many disorders. Here, we aim to explore if genetic variants in MST2 are associated with the risk of non-syndromic cleft lip with or without palate (NSCL/P). Materials and methodsThe association study was performed in a two-stage study of 1,069 cases and 1,724 controls to evaluate the association between genetic variants in the MST2 and NSCL/P risk. The potential function of the candidate single nucleotide polymorphism (SNP) was predicted using HaploReg, RegulomeDB, and public craniofacial histone chromatin immunoprecipitation sequencing (ChIP-seq) data. Haploview was used to perform the haplotype of risk alleles. The expression quantitative trait loci (eQTL) effect was assessed using the Genotype-Tissue Expression (GTEx) project. Gene expression in mouse embryo tissue was performed using data downloaded from GSE67985. The potential role of candidate gene in the development of NSCL/P was assessed by correlation and enrichment analysis. ResultsAmong SNPs in MST2, rs2922070 C allele (Pmeta = 2.93E-04) and rs6988087 T allele (Pmeta = 1.57E-03) were linked with significantly increased risk of NSCL/P. Rs2922070, rs6988087 and their high linkage disequilibrium (LD) SNPs constituted a risk haplotype of NSCL/P. Individuals carrying 3–4 risk alleles had an elevated risk of NSCL/P compared to those who carried less risk alleles (P = 2.00E-04). The eQTL analysis revealed a significant association between these two variants and MST2 in muscle tissue of the body. The MST2 expressed during mouse craniofacial development and over-expressed in the human orbicularis oris muscle (OOM) of NSCL/P patients compared to controls. MST2 was involved in the development of NSCL/P by regulating the mRNA surveillance pathway, the MAPK signaling pathway, the neurotrophin signaling pathway, the FoxO signaling pathway and the VEGF signaling pathway. ConclusionMST2 was associated with the development of NSCL/P.

Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

The low survival rate of mesenchymal stem cells (MSCs) caused by anoikis, a form of apoptosis, limits the therapeutic efficacy of MSCs. As a proapoptotic molecule, mammalian Ste20-like kinase 1 (Mst1) can increase the production of reactive oxygen species (ROS), thereby promoting anoikis. Recently, we found that Mst1 inhibition could protect mouse bone marrow MSCs (mBMSCs) from H2O2-induced cell apoptosis by inducing autophagy and reducing ROS production. However, the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear. To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs. Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA (shRNA) adenovirus transfection. Integrin (ITGs) were tested by flow cytometry. Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA, respectively. The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays. The levels of the anoikis-related proteins ITGα5, ITGβ1, and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I, Beclin1 and p62 were detected by Western blotting. In isolated mBMSCs, Mst1 expression was upregulated, and Mst1 inhibition significantly reduced cell apoptosis, induced autophagy and decreased ROS levels. Mechanistically, we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4, ITGαv, or ITGβ3 expression. Moreover, autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis. Mst1 inhibition ameliorated autophagy formation, increased ITGα5β1 expression, and decreased the excessive production of ROS, thereby reducing cell apoptosis in isolated mBMSCs. Based on these results, Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

Sterile 20-like kinase 3 promotes tick-borne encephalitis virus assembly by interacting with NS2A and prM and enhancing the NS2A-NS4A association.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.

Activation of Hippo Pathway Proteins in Thrombin-Stimulated Platelets and Regutates Platelets Activation in Vitro

To investigate the phosphorylation levels of Hippo pathway proteins in thrombin stimulated platelets and to explore its effects on platelet activation. The phosphorylation levels of Hippo pathway proteins - Mammalian STE20-like kinase 1/2 (MST1/2), Nuclear Dbf2 related kinase 1/2 (NDR1/2) and Mps one binder 1(MOB1) in human thrombin stimulated platelets was detected by Western blot. The effect of MST1/2 inhibitor XMU-MP-1 on platelet aggregation was detected by Platelet Aggregometer. The effect of XMU-MP-1 on platelet integrin αIIbβ3 activation and CD62p expression was detected by flow cytometry. The effect of XMU-MP-1 on the "outside-in" signal of platelet integrin was detected by blood clot retraction test. The effects of XMU-MP-1 on platelet Hippo pathway proteins and p38 phosphorylation levels was detected by Western blot. The phosphorylation levels of MST1/2, NDR1/2 and MOB1 were significantly increased in thrombin activated platelets; XMU-MP-1 inhibited thrombin-induced platelet aggregation and αIIbβ3 activation, but did not affect α-granules release and clot retraction. In addition, thrombin induced phosphorylation of the Hippo proteins were decreased in XMU-MP-1 treated platelets, while the phosphorylation of p38 was not affected. In thrombin stimulated platelets, Hippo pathway proteins were activated and contributed to platelets activation.

Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

The cytosolic viral nucleic acid-sensing pathways converge on the protein kinase TANK-binding kinase 1 (TBK1) and the transcription factor interferon (IFN)-regulatory factor 3 (IRF3) to induce type I IFN production and antiviral immune responses. However, the mechanism that triggers the binding of TBK1 and IRF3 after virus infection remains not fully understood. Here, we identified that thousand and one kinase 1 (TAOK1), a Ste20-like kinase, positively regulated virus-induced antiviral immune responses by controlling the TBK1-IRF3 signaling axis. Virus invasion downregulated the expression of TAOK1. TAOK1 deficiency resulted in decreased nucleic acid-mediated type I IFN production and increased susceptibility to virus infection. TAOK1 was constitutively associated with TBK1 independently of the mitochondrial antiviral signaling protein MAVS. TAOK1 promoted IRF3 activation by enhancing TBK1-IRF3 complex formation. TAOK1 enhanced virus-induced type I IFN production in a kinase activity-dependent manner. Viral infection induced TAOK1 to bind with dynein instead of microtubule-associated protein 4 (MAP4), leading to the trafficking of TBK1 to the perinuclear region to bind IRF3. Thus, the depolymerization of microtubule impaired virus-mediated IRF3 activation. Our results revealed that TAOK1 functioned as a new interaction partner and regulated antiviral signaling via trafficking TBK1 along microtubules to bind IRF3. These findings provided novel insights into the function of TAOK1 in the antiviral innate immune response and its related clinical significance.

Ste20-like kinase activity promotes meiotic resumption and spindle microtubule stability in mouse oocytes.

Ste20-like kinase (SLK) is involved in cell proliferation and migration in somatic cells. This study aims to explore SLK expression and function in mouse oocyte meiosis. Western blot, immunofluorescence, Co-immunoprecipitation, drug treatment, cRNA construct and in vitro transcription, microinjection of morpholino oilgo (MO) and cRNA were performed in oocytes. High and stable protein expression of SLK was detected in mouse oocyte meiosis, with dynamic distribution in the nucleus, chromosomes and spindle apparatus. SLK phosphorylation emerges around meiotic resumption and reaches a peak during metaphase I (MI) and metaphase II. SLK knockdown with MO or expression of kinase-dead SLK K63R dramatically delays meiotic resumption due to sequentially suppressed phosphorylation of Polo-like kinase 1 (Plk1) and cell division cycle 25C (CDC25C) and dephosphorylation of cyclin-dependent kinase 1 (CDK1). SLK depletion promotes ubiquitination-mediated degradation of paxillin, an antagonist to α-tubulin deacetylation, and thus destroys spindle assembly and chromosome alignment; these phenotypes can be substantially rescued by exogenous expression of SLK kinase active fragment. Additionally, exogenous SLK effectively promotes meiotic progression and spindle assembly in aging oocytes with reduced SLK. Collectively, this study reveals SLK is required for meiotic resumption and spindle assembly in mouse oocyte meiosis.

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase.

Maintaining an appropriate balance between excitation and inhibition is critical for neuronal information processing. Cortical neurons can cell-autonomously adjust the inhibition they receive to individual levels of excitatory input, but the underlying mechanisms are unclear. We describe that Ste20-like kinase (SLK) mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feedforward circuit, but not in the feedback circuit. This effect is due to regulation of inhibition originating from parvalbumin-expressing interneurons, while inhibition via somatostatin-expressing interneurons is unaffected. Computational modeling shows that this mechanism promotes stable excitatory-inhibitory ratios across pyramidal cells and ensures robust and sparse coding. Patch-clamp RNA sequencing yields genes differentially regulated by SLK knockdown, as well as genes associated with excitation-inhibition balance participating in transsynaptic communication and cytoskeletal dynamics. These data identify a mechanism for cell-autonomous regulation of a specific inhibitory circuit that is critical to ensure that a majority of cortical pyramidal cells participate in information coding.

Mechanotransduction in the pathogenesis of non-alcoholic fatty liver disease.

Mechanobiology is a domain of interdisciplinary research that aims to explore the impact of physical force, applied externally or internally, on cell and tissue function, including development, growth, and differentiation. Mechanotransduction is a term that describes how cells sense physical forces (such as compression, stretch, and shear stress), convert them into biochemical signals, and mount adaptive responses integrated by the nucleus. There is accumulating evidence that mechanical forces extensively inform the biological behaviour of liver cells in health and disease. Recent research has elucidated many cellular and molecular mechanisms involved in this process including the pleiotropic control and diverse effects of the paralogous transcription co-activators YAP/TAZ, which play a prominent role in mechanotransduction. The liver sinusoids represent a unique microenvironment in which cells are exposed to mechanical cues originating in the cytoskeleton and at interfaces with adjacent cells, the extracellular matrix, and vascular or interstitial fluids. In non-alcoholic fatty liver disease (NAFLD), hepatocellular lipid accumulation and ballooning, activation of inflammatory responses, dysfunction of liver sinusoidal endothelial cells, and transdifferentiation of hepatic stellate cells into a pro-contractile and pro-fibrotic phenotype have been associated with aberrant cycles of mechanosensing and mechanoresponses. The downstream consequences of disrupted mechanical homeostasis likely contribute to the progression of NAFLD and promote the development of portal hypertension, cirrhosis, and hepatocellular carcinoma. Identification of molecular targets involved in pathogenic mechanotransduction will allow for the development of novel strategies to prevent the progression of liver disease in NAFLD.

MST4 as a novel therapeutic target for autophagy and radiosensitivity in gastric cancer.

Mammalian ste20-like kinase 4 (MST4) and autophagy have been implicated in ailments such as inflammatory and cancers. In this study, the expression of MST4 data was extracted from TCGA, GTEx, and GEPIA. The infiltration of immune cells and methylation level of MST4 in tumor and normal tissues were extracted from GEPIA 2021, TISIDB, UALCAN, EWAS, MethSurv, and MEXPRESS database. We also predict the efficacy of outcome prediction with receiver operating characteristic curve (ROC). All proteins expressions of MST4, P62, and LC3 were detected by immunohistochemistry (IHC) in paired Gastric cancer (GC) and para-cancerous normal tissue samples. We verify the effects of MST4 on irradiation-induced gastric death, and also investigate effects of MST4 activating autophagy in GC cell lines with various in vitro assays using western blotting. We have confirmed the high transcription level of MST4 from TCGA, USLCAN, HPA, and other portals, but a rapid decrease in protein level. More, MST4 can be considered as an independent prognostic molecule, which has significant prognostic significance in tumor grade, anti-tumor treatment, histological type, and time-dependent ROC curve. The methylation degree of MST4 promoter region in tumor is much lower than that in normal tissue, which may be the main reason for the remarkably high transcription level of MST4. In addition, MST4 transcription level was significantly inversely proportional to the infiltration level of most immune cells. The MST4 up-regulation and the positive association of MST4 with autophagy expression were cross-validated in open-access datasets. MST4, as an autophagy-associated protein, plays a potential role in inducing cell death by increasing protein content in radiotherapy.

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.

Effect of Inactivation of Mst1 and Mst2 in the Mouse Adrenal Cortex.

Recent conditional knockout of core components of the Hippo signaling pathway in the adrenal gland of mice has demonstrated that this pathway must be tightly regulated to ensure proper development and maintenance of the adrenal cortex. We report herein that the most upstream kinases of the pathway, the mammalian STE20-like protein kinases 1 and 2 (MST1and MST2, respectively), are expressed in the mouse adrenal cortex with MST2 expression being restricted to the zona glomerulosa (zG). To further explore the role of Hippo signaling in adrenocortical cells, we conditionally deleted Mst1/2 in steroidogenic cells using an Nr5a1-cre strain (Mst1 flox/flox ; Mst2 flox/flox ; Nr5a1-cre). Our results show that the loss of MST1/2 leads to the premature and progressive accumulation of subcapsular GATA4+, WT1+ adrenal gonadal primordium (AGP)-like progenitor cells starting at 2 months of age without affecting aldosterone and corticosterone secretion. To help us understand this phenotype, microarray analyses were performed on adrenal glands from 2-month-old mutant and control mice. Gene expression analyses revealed that loss of Mst1/2 leads to the overexpression of known downstream target genes (Ajuba, Aqp1, Fn1, Ibsp, Igf1, Igfbp2, Mmp2, Thbs1) of the main effector of Hippo signaling, YAP; and underexpression of genes (Agtr1b, Ecgr4, Hsd3b6, Nr0b1, Tesc, Vsnl1) that are normally specifically expressed in the zG or overexpressed in the zG compared to the zona fasciculata (zF). Together, these results suggest that MST1/2 regulates Hippo signaling activity in the adrenal cortex and that these two kinases are also involved in the fine tuning of zG cell function or differentiation.

The evolutionarily highly conserved MST2 suppresses WSSV infection by promoting apoptosis in shrimp

Mammalian Ste20-like kinase 2 (MST2) is a core component of the Hippo pathway and plays an important role in regulating cell apoptosis, cell proliferation and cell cycle progression, but it has rarely been studied in invertebrates, especially crustaceans. In this study, the MST2 gene in penaeus japonicus shrimp was characterized. A 2111 bp long nucleotide sequence encoding 690 amino acids was obtained. The molecular weight of MST2 protein is 78.47222 kDa. The bioinformatics analysis indicated that the MST2 protein has an S_TK-c (Ser/Thr kinase) domain and a SARAH (Salvador/Ras-association domain family/Hippo) domain, which is consistent with other species. The analysis of subcellular localization showed that MST2 was mainly present in the cytoplasm. Real time PCR and apoptosis analysis indicated that MST2 was involved in the white spot syndrome virus (WSSV) infection process and inhibited viral infection by promoting apoptosis. This study revealed the potential function of MST2 in virus invasion and cell apoptosis, providing a new target for preventing shrimp virus infection.

GATA3 positively regulates PAR1 to facilitate in vitro disease progression and decrease cisplatin sensitivity in neuroblastoma via inhibiting the hippo pathway.

GATA binding protein 3 (GATA3) is reported to critically involved in the pathogenesis of neuroblastoma (NB). This study investigated the specific role and mechanism of GATA3 in NB progression. JASPAR was utilized to predict GATA3's downstream targets, whose binding relation with GATA3 was inspected by a dual-luciferase reporter assay. NB cell lines underwent transfection of GATA3/protease-activated receptor 1 (PAR1) overexpression plasmids or shGATA3, followed by cisplatin treatment. NB cell sensitivity to cisplatin, viability, migration, invasion, cell cycle progression and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing assay, transwell assay and flow cytometry, respectively. Expressions of GATA3, PAR1, epithelial-mesenchymal transition-related molecules (N-Cadherin and Vimentin), hippo pathway-related molecules (mammalian Ste20-like kinase (Mst)1, Mst2, Mps one binding (Mob) 1, phosphorylated (p)-Mob1, Yes-associated protein (YAP) and p-YAP) in NB tissues and cell lines were assessed by western blot or qRT-PCR. GATA3 expression was increased in NB tissues and cells. GATA3 overexpression increased NB cell viability, promoted migration, invasion, and cell cycle progression, increased the expressions of N-Cadherin, Vimentin and YAP, decreased the expressions of Mst1, Mst2, Mob1, p-Mob1, p-YAP and the ratio of p-YAP to YAP, and attenuated cisplatin-induce cell apoptosis, which GATA3 knockdown induced the opposite effect. GATA3 directly targeted PAR1, whose overexpression increased NB cell viability, inhibited the hippo pathway, and attenuated cisplatin-induce cell apoptosis, and reversed GATA3 knockdown-induced effect on these aspects. GATA3 positively regulates PAR1 to facilitate in-vitro disease progression and decrease cisplatin sensitivity in NB via inhibiting the hippo pathway.

Retraction: Mammalian STE20-like kinase 2 promotes lipopolysaccharides-mediated cardiomyocyte inflammation and apoptosis by enhancing mitochondrial fission

The Publisher retracts the cited article. Following publication, the publisher uncovered evidence that false identities were used in the peer-review process. The assignment of a fake reviewer was confirmed by an investigation, conducted in accordance with Frontiers’ policies and the Committee on Publication Ethics (COPE) guidelines. The investigation also uncovered concerns about the presentation and validity of the data in the article that normally would have led to a rejection. When contacted, the authors failed to provide a data set that adequately supports the reported conclusions. The authors do not agree to this retraction. This retraction was approved by the Chief Editors of Frontiers in Physiology and the Chief Executive Editor of Frontiers.

Evaluation of the significance of human class I homeobox A13 and STK4/MST1 expression in conventional colorectal adenoma and adenocarcinoma and correlation with clinicopathological parameters (immunohistochemical study)

Background Colorectal carcinoma (CRC) is the third most common cancer in the world. Human class I homeobox A13 (HOXA13) was initially identified as a transcription factor and has an important role in embryonic development and malignant transformation. Mammalian STE20-like kinase 1 (STK4/MST1) encodes a serine/threonine kinase that is the mammalian homolog of the Hippo pathway and plays an important role in controlling cell growth, apoptosis, and organ size. Aim This work aimed to evaluate the role of HOXA13 and STK4/MST1 in conventional colorectal adenoma and adenocarcinoma. Materials and methods This retrospective study was carried out on 20 cases of conventional colorectal adenoma and 30 cases of conventional colorectal adenocarcinoma. HOXA13 and STK4/MST1 immunostaining was done and assessed for each case. Correlation with the clinicopathological findings and statistical analysis was studied. Results In the carcinoma cases, there was a highly significant direct statistical correlation between HOXA13 expression and tumor grade, lymph node metastasis, and TNM stage (P<0.01 for each). Inverse statistical correlation between STK4/MST1 expression and tumor grade, depth of tumor invasion (T), and TNM stage (P<0.01 for each) was found. HOXA13 and STK4/MST1 immunoexpression showed direct highly significant relation with the transition from adenoma to adenoma with dysplasia to adenocarcinoma (P<0.01). There was a highly significant inverse statistical correlation between HOXA13 and STK4/MST1 expression in the studied cases of CRC (P<0.01). According to receiver-operating characteristic curve, both markers were good in the prediction of metastatic potential in the carcinoma cases using the TNM stage as a parameter with a sensitivity of 85%. Conclusion HOXA13 was immunohistochemically overexpressed, while STK4/MST1 was downexpressed in progression from colorectal adenoma to adenoma with dysplasia to adenocarcinoma cases. HOXA13 and STK4/MST1 might have a potential role as independent prognostic factors in CRC and may have validity to predict metastatic potential of CRC.

Combinatorial targeting of Hippo-STRIPAK and PARP elicits synthetic lethality in gastrointestinal cancers.

The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli that result in intracellular activities. Previously, we discovered that STRIPAK is a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex as an essential player in the control of DNA double-stranded break (DSB) repair and genomic stability. Specifically, we found that the mammalian STE20-like protein kinases 1 and 2 (MST1/2), independent of classical Hippo signaling, directly phosphorylated zinc finger MYND type–containing 8 (ZMYND8) and hence resulted in the suppression of DNA repair in the nucleus. In response to genotoxic stress, the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via TANK-binding kinase 1–induced (TBK1-induced) structural stabilization of the suppressor of IKBKE 1– sarcolemma membrane–associated protein (SIKE1-SLMAP) arm. As such, we found that STRIPAK-mediated MST1/2 inactivation increased the DSB repair capacity of cancer cells and endowed these cells with resistance to radio- and chemotherapy and poly(ADP-ribose)polymerase (PARP) inhibition. Importantly, targeting the STRIPAK assembly with each of 3 distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and resensitize cancer cells to PARP inhibitors in both animal- and patient-derived tumor models. Overall, our findings not only uncover what we believe to be a previously unrecognized role for STRIPAK in modulating DSB repair but also provide translational implications of cotargeting STRIPAK and PARP for a new type of synthetic lethality anticancer therapy.

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.

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics

We have recently purified mammalian sterile 20 (STE20)–like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase–related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca2+/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.

Activation of YAP1 by STK25 contributes to the progression of hepatocellular carcinoma

A pathogenic role of serine/threonine protein kinase 25 (STK25) has been observed in several chronic liver diseases. However, whether STK25 participates in hepatocellular carcinoma (HCC) remains unexplored. The current work aimed to explore the role and mechanism of STK25 in HCC. A high STK25 level was found in HCC tissue, which was associated with reduced overall survival. HCC cells with STK25 silencing displayed a marked decrease in proliferative and invasive ability, but were highly sensitive to apoptosis induced by the chemotherapy drug sorafenib. Reciprocally, HCC cells with forced expression of STK25 displayed the opposite effects. Further data unveiled that STK25 silencing restrained the activation of Yes-associated protein 1 (YAP1) associated with regulation of mammalian STE20-like protein kinase 1/2 (MST1/2). Forced expression of constitutively active YAP1 abolished STK25 silencing-induced antitumor effects, while repression of YAP1 reversed STK25 upregulation-induced protumor effects. Additionally, HCC cells with STK25 silencing exhibited reduced tumorigenic potential in vivo. Collectively, the results show that STK25 exerts a protumor function in HCC by enhancing YAP1 activation via regulation of MST1/2. These findings propose STK25 as a viable target for the development of anti-HCC therapy.