Hippo and PI5P4K signaling intersect to control the transcriptional activation of YAP.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Regulatory role of PDK1 via integrated gene analysis of mitochondria-immune response in periodontitis.

AIMS: To investigate the association between mitochondrial events and immune response in periodontitis and related regulatory genes. MAIN METHODS: Gene expression profiles in gingival tissues were retrieved from the Gene Expression Omnibus. Mitochondria-immune response-related differentially expressed genes (MIR-DEGs) between the healthy and periodontitis samples were determined. WGCNA, GO, and KEGG were used to investigate the function and the enriched pathways of MIR-DEGs. The correlation between MIR-DEGs expression and clinical probing pocket depth was analyzed. The MIR-DEGs were further identified and verified in animal samples. A periodontitis model was established in C57BL/6 mice with silk ligation. Micro-computed tomography was used to assess alveolar bone loss. Western blot, quantitative real-time polymerase chain reaction, and immunohistochemical analyses further validated the differential expression of the MIR-DEGs. KEY FINDINGS: A total of ten MIR-DEGs (CYP24A1, PRDX4, GLDC, PDK1, BCL2A1, CBR3, ARMCX3, BNIP3, IFI27, and UNG) were identified, the expression of which could effectively distinguish patients with periodontitis from the healthy controls. Enhanced immune response was detected in the periodontitis group with that in the healthy controls, especially in B cells. PDK1 was a critical MIR-DEG correlated with B cell immune response and clinical periodontal probing pocket depth. Both animal and clinical periodontal samples presented higher gene and protein expression of PDK1 than the control samples. Additionally, PDK1 colocalized with B cells in both animal and clinical periodontal tissues. SIGNIFICANCE: Mitochondria participate in the regulation of the immune response in periodontitis. PDK1 may be the key mitochondria-related gene regulating B-cell immune response in periodontitis.

The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

The Mst1/2-BNIP3 axis is required for mitophagy induction and neuronal viability under mitochondrial stress.

Mitophagy induction upon mitochondrial stress is critical for maintaining mitochondrial homeostasis and cellular function. Here, we found that Mst1/2 (Stk3/4), key regulators of the Hippo pathway, are required for the induction of mitophagy under various mitochondrial stress conditions. Knockdown of Mst1/2 or pharmacological inhibition by XMU-MP-1 treatment led to impaired mitophagy induction upon CCCP and DFP treatment. Mechanistically, Mst1/2 induces mitophagy independently of the PINK1-Parkin pathway and the canonical Hippo pathway. Moreover, our results suggest the essential involvement of BNIP3 in Mst1/2-mediated mitophagy induction upon mitochondrial stress. Notably, Mst1/2 knockdown diminishes mitophagy induction, exacerbates mitochondrial dysfunction, and reduces cellular survival upon neurotoxic stress in both SH-SY5Y cells and Drosophila models. Conversely, Mst1 and Mst2 expression enhances mitophagy induction and cell survival. In addition, AAV-mediated Mst1 expression reduced the loss of TH-positive neurons, ameliorated behavioral deficits, and improved mitochondrial function in an MPTP-induced Parkinson's disease mouse model. Our findings reveal the Mst1/2-BNIP3 regulatory axis as a novel mediator of mitophagy induction under conditions of mitochondrial stress and suggest that Mst1/2 play a pivotal role in maintaining mitochondrial function and neuronal viability in response to neurotoxic treatment.

Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury.

The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout (dKO) of the upstream Hippo kinases Mst1 and Mst2 promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell overproliferation, tubular injury, and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistent with this, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell overproliferation, tubular injury, and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, where tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell overproliferation, tubular injury, and renal inflammation.

MST1/2: Important regulators of Hippo pathway in immune system associated diseases.

The Hippo signaling pathway is first found in Drosophila and is highly conserved in evolution. Previous studies on this pathway in mammals have revealed its key role in cell proliferation and differentiation, organ size control, and carcinogenesis. Apart from these, recent findings indicate that mammalian Ste20-like kinases 1 and 2 (MST1/2) have significant effects on immune regulation. In this review, we summarize the updated understanding of how MST1/2 affect the regulation of the immune system and the specific mechanism. The effect of MST1/2 on immune cells and its role in the tumor immune microenvironment can alter the body's response to tumor cells. The relationship between MST1/2 and the immune system suggests new directions in the manipulation of immune responses for clinical immunotherapy, especially for tumor treatment.

Association Study between SNPs in MST1 and MST2 and H. pylori Infection as well as Noncardia Gastric Carcinogenesis.

INTRODUCTION: Gastric cancer (GC) remains a global health challenge, and H. pylori infection is a main risk factor for noncardia GC. The present study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in mammalian sterile 20-like kinase 1 (MST1) and MST2, H. pylori (H. pylori) infection, and the risk of noncardia gastric cancer (GC). METHODS: A case-control study was conducted using enzyme-linked immunosorbent assay (ELISA) and TaqMan method to detect the titer of anti-H. pylori antibody in normal human serum and genotype 9 SNPs of MST1 and MST2 genes among 808 samples. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between SNPs and H. pylori infection, as well as the risk of noncardia gastric cancer in codominant, dominant, overdominant, recessive, and log-additive genetic models. Haplotypes were constructed using the Haploview 4.2 software. RESULTS: The CC genotype of MST2 SNP rs10955176 was associated with a reduced risk of H. pylori infection compared to the TT + CT genotype. None of other SNPs were associated with H. pylori infection. The TT genotype of MST2 SNP rs7827435 was associated with a reduced risk of noncardia gastric cancer compared to the AA + AT genotype. None of the SNPs were associated with noncardia gastric cancer. There were no associations between haplotypes and H. pylori infection or the risk of noncardia gastric cancer. CONCLUSIONS: The CC genotype of rs10955176 and the TT genotype of rs7827435 may serve as protective factors against H. pylori infection and noncardia gastric cancer risk, respectively.

MAP4K2 connects the Hippo pathway to autophagy in response to energy stress.

As a key regulator of development, organ size, tissue homeostasis and cancer, the Hippo pathway is tightly regulated by various growth-related signaling events. Among them, energy stress activates the Hippo pathway to inhibit its downstream effector YAP1. Our recent work reported a YAP1-independent role of the Hippo pathway in promoting macroautophagy/autophagy and cell survival in response to energy stress, a process mediated by Hippo kinase MAP4K2. MAP4K2 interacts with and phosphorylates MAP1LC3A/LC3A at S87, which in turn drives autophagosome-lysosome fusion via the RAB3GAP-RAB18 axis. Energy stress activates MAP4K2 by reducing its association with the Hippo phosphatase complex STRIPAK component STRN4. Moreover, MAP4K2 is highly expressed in head and neck cancer, while MAP4K2 and its mediated autophagy are required for head and neck cancer development. Taken together, our study not only reveals a noncanonical role of the Hippo pathway in energy stress response, but also suggests Hippo kinase MAP4K2 as a potential therapeutic target for head and neck cancer treatment.Abbreviation: AMPK: 5'-AMP-activated protein kinase; Atg8: autophagy related 8; LATS1: large tumor suppressor 1; LIR: microtubule-associated protein 1 light chain 3-interacting region; MAP1LC3A/LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP4K2: mitogen-activated protein kinase kinase kinase kinase 2; PPP2/PP2A: protein phosphatase 2; RAB3GAP: RAB3 GTPase activating protein; RAB18: RAB18, member RAS oncogene family; SLMAP: sarcolemma associated protein; STK3/MST2: serine/threonine kinase 3; STK4/MST1: serine/threonine kinase 4; STRIPAK: striatin-interacting phosphatase and kinase; STRN4: striatin, calmodulin binding protein 4; SQSTM1/p62: sequestosome 1; TEAD: TEA domain family member; ULK1: unc-51 like kinase 1; WWTR1/TAZ: WW domain containing transcription regulator 1; YAP1: yes-associated protein 1.

Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

MST2 methylation by PRMT5 inhibits Hippo signaling and promotes pancreatic cancer progression.

The Hippo signaling axis is a tumor suppressor pathway that is activated by various extra-pathway factors to regulate cell differentiation and organ development. Recent studies have reported that autophosphorylation of the core kinase cassette stimulates activation of the Hippo signaling cascade. Here, we demonstrate that protein arginine methyltransferase 5 (PRMT5) contributes to inactivation of the Hippo signaling pathway in pancreatic cancer. We show that the Hippo pathway initiator serine/threonine kinase 3 (STK3, also known as MST2) of Hippo signaling pathway can be symmetrically di-methylated by PRMT5 at arginine-461 (R461) and arginine-467 (R467) in its SARAH domain. Methylation suppresses MST2 autophosphorylation and kinase activity by blocking its homodimerization, thereby inactivating Hippo signaling pathway in pancreatic cancer. Moreover, we also show that the specific PRMT5 inhibitor GSK3326595 re-activates the dysregulated Hippo signaling pathway and inhibits the growth of human pancreatic cancer xenografts in immunodeficient mice, thus suggesting potential clinical application of PRMT5 inhibitors in pancreatic cancer.

TRIMming away colon cancer: TRIM21-mediated ubiquitination as an activator of the Hippo tumor suppressor pathway.

In this issue of Cell Chemical Biology, Liu et al.1 identify TRIM21-mediated ubiquitination of the Hippo pathway kinase MST2, promoting its dimerization and activation. The antidepressant Vilazodone was found to bind to TRIM21, enhancing its activity toward MST2, increasing Hippo activation, and reducing colorectal cancer metastasis.

Serine/threonine kinase 3 promotes oxidative stress and mitochondrial damage in septic cardiomyopathy through inducing Kelch-like ECH-associated protein 1 phosphorylation and nuclear factor erythroid 2-related factor 2 degradation.

Serine/threonine kinases (STK3) is a core component of the Hippo pathway and modulates oxidative stress and inflammatory responses in cardiovascular diseases. However, its potential role in septic cardiomyopathy remains undefined. STK3-mediated phosphorylation of Kelch-like ECH-associated protein 1 (KEAP1) was shown to suppress antioxidant gene transcription controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages. To explore whether STK3 induces KEAP1-mediated suppression of Nrf2 in septic cardiomyopathy, wild-type and global STK3 knockout (STK3 -/- ) mice were treated with LPS. LPS treatment upregulated cardiac STK3 expression. STK3 deletion attenuated myocardial inflammation and cardiomyocyte death, and improved myocardial structure and function. In LPS-challenged HL-1 cardiomyocytes, shRNA-mediated STK3 knockdown normalized mitochondrial membrane potential and ATP production, attenuated apoptosis, and rescued antioxidant gene expression by preventing Nrf2 downregulation. Co-IP, docking analysis, western blotting, and immunofluorescence assays further showed that STK3 binds and phosphorylates KEAP1, promoting Nrf2 downregulation. Accordingly, transfection of phosphodefective KEAP1 mutant protein in cardiomyocyte restored Nrf2 expression and mitochondrial performance upon LPS, while expression of a phosphomimetic KEAP1 mutant abolished the mitochondria-protective and pro-survival effects of STK3 deletion. These findings suggest that STK3 upregulation contributes to septic cardiomyopathy by phosphorylating KEAP1 to promote Nrf2 degradation and suppression of the antioxidant response.

Effect of STK3 on proliferation and apoptosis of pancreatic cancer cells via PI3K/AKT/mTOR pathway.

Pancreatic cancer, as a malignant tumor with a very poor prognosis, has a high mortality. It is imperative to clarify the mechanism of pancreatic cancer development and find suitable targets for diagnosis and treatment. Serine/threonine kinase 3 (STK3) is one of the core kinases of the Hippo pathway and has the ability to inhibit tumor growth. But the biological function of STK3 in pancreatic cancer remains unknown. Here, we confirmed that STK3 has an impact on the growth, apoptosis, and metastasis of pancreatic cancer cells and investigated the related molecular mechanisms. In our research, we found that STK3 is reduced in pancreatic cancer by RT-qPCR, IHC and IF, its expression level is correlated with the clinicopathological features. CCK-8 assay, colony formation assay and flow cytometry were used to detect the effect of STK3 on the proliferation and apoptosis of pancreatic cancer cells. In addition, the Transwell assay was used to detect the ability of cell migration and invasion. The results showed that STK3 promoted apoptosis and inhibited cell migration, invasion and proliferation in pancreatic cancer. Gene set enrichment analysis (GSEA) and western blotting are used to predict and verify the pathways related to STK3. Subsequently, we found that the effect of STK3 on proliferation and apoptosis is closely related to the PI3K/AKT/mTOR pathway. Moreover, the assistance of RASSF1 plays a significant role in the regulation of PI3K/AKT/mTOR pathway by STK3. The nude mouse xenograft experiment demonstrated the tumor suppressive ability of STK3 in vivo. Collectively, this study found that STK3 regulates pancreatic cancer cell proliferation and apoptosis by suppressing the PI3K/AKT/mTOR pathway with the assistance of RASSF1.

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.

BACKGROUND: Appropriate 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. OBJECTIVES: Our 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. METHODS: Forty 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. RESULTS: SAT 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. CONCLUSIONS: Obesity 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.

Extracellular matrix stiffness regulates degradation of MST2 via SCF ßTrCP.

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

Interaction of LATS1 with SMAC links the MST2/Hippo pathway with apoptosis in an IAP-dependent manner.

Metastatic malignant melanoma is the deadliest skin cancer, and it is characterised by its high resistance to apoptosis. The main melanoma driving mutations are part of ERK pathway, with BRAF mutations being the most frequent ones, followed by NRAS, NF1 and MEK mutations. Increasing evidence shows that the MST2/Hippo pathway is also deregulated in melanoma. While mutations are rare, MST2/Hippo pathway core proteins expression levels are often dysregulated in melanoma. The expression of the tumour suppressor RASSF1A, a bona fide activator of the MST2 pathway, is silenced by promoter methylation in over half of melanomas and correlates with poor prognosis. Here, using mass spectrometry-based interaction proteomics we identified the Second Mitochondria-derived Activator of Caspases (SMAC) as a novel LATS1 interactor. We show that RASSF1A-dependent activation of the MST2 pathway promotes LATS1-SMAC interaction and negatively regulates the antiapoptotic signal mediated by the members of the IAP family. Moreover, proteomic experiments identified a common cluster of apoptotic regulators that bind to SMAC and LATS1. Mechanistic analysis shows that the LATS1-SMAC complex promotes XIAP ubiquitination and its subsequent degradation which ultimately results in apoptosis. Importantly, we show that the oncogenic BRAFV600E mutant prevents the proapoptotic signal mediated by the LATS1-SMAC complex while treatment of melanoma cell lines with BRAF inhibitors promotes the formation of this complex, indicating that inhibition of the LATS1-SMAC might be necessary for BRAFV600E-driven melanoma. Finally, we show that LATS1-SMAC interaction is regulated by the SMAC mimetic Birinapant, which requires C-IAP1 inhibition and the degradation of XIAP, suggesting that the MST2 pathway is part of the mechanism of action of Birinapant. Overall, the current work shows that SMAC-dependent apoptosis is regulated by the LATS1 tumour suppressor and supports the idea that LATS1 is a signalling hub that regulates the crosstalk between the MST2 pathway, the apoptotic network and the ERK pathway.

High MST2 expression regulates lens epithelial cell apoptosis in age-related cataracts through YAP1 targeting GLUT1.

Age-related cataract (ARC) is a severe visual impairment disease and its pathogenesis remains unclear. This study investigated the relevance of MST2/YAP1/GLUT1 in ARC development in vivo and in vitro, and explored the role and possible mechanisms of this pathway in oxidative damage-mediated apoptosis of lens epithelial cells (LECs). Western blot analysis and immunohistochemistry showed that MST2 and phosphorylated (p)-YAP (Ser127) protein levels were increased, and YAP1 and GLUT1 protein levels were decreased in LECs of ARC patients and aged mice. Additionally, differential expression of MST2 and YAP1 was associated with H2O2-induced apoptosis of human lens epithelial B3 (HLE-B3) cells. CCK-8 and Hoechst 33,342 apoptosis assays showed that MST2 and YAP1 were involved in H2O2-induced apoptosis of LECs. Subsequent experiments showed that, during MST2-mediated H2O2-induced apoptosis, p-YAP (Ser127) levels were elevated and immunofluorescence revealed nucleoplasmic translocation and inhibition of YAP1 protein expression. Furthermore, GLUT1 was in turn synergistically transcriptionally regulated by YAP1-TEAD1 in dual luciferase reporter assays. In conclusion, our study indicates that the MST2/YAP1/GLUT1 pathway plays a major role in the pathogenesis of ARC and LECs apoptosis, providing a new direction for future development of targeted inhibitors that block this signaling pathway to prevent, delay, or even cure ARC.

circHIPK3 regulates cell proliferation and migration by sponging microRNA-124 and regulating serine/threonine kinase 3 expression in esophageal squamous cell carcinoma.

Circular RNAs (circRNAs) are a type of important non-coding RNAs that widely involve in the physiological and pathophysiological process. Recent research has established a link between circHIPK3 and the malignant activity of cancer cells. However, circHIPK3' role in esophageal squamous cell carcinoma (ESCC) still needs more focus. To determine the prognostic value of circHIPK3 in patients with ESCC, the expression of circHIPK3 was quantified in 32 pairs of ESCC using real-time polymerase chain reaction (RT-qPCR). Then, the correlation between circHIPK3 expression and clinical characteristics of patients was also analyzed. The function of circHIPK3 in the development of ESCC was investigated using cell biology studies and bioinformatics. The results showed that the expression of circHIPK3 was considerably higher in tumor tissues from ESCC patients than that of adjacent tissues, which was associated with a poor prognosis. Additionally, silencing of circHIPK3 expression retarded esophageal cancer cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, as well as the growth in vivo. Mechanistically, we discovered that circHIPK3 behaved like a sponge, absorbing microRNA-124 (miR-124) and promoting serine/threonine kinase 3 (AKT3) expression. Our findings indicate that circHIPK3 acts as an oncogene in ESCC and that the circHIPK3-AKT3 axis may be a therapeutic target for patients with ESCC.

MST1/2 in PDGFRα+ cells negatively regulates TGF-ß-induced myofibroblast accumulation in renal fibrosis.

Injury-induced fibroblast-to-myofibroblast differentiation is a key event of renal fibrosis. Yes-associated protein (YAP), a transcriptional coactivator, plays an important role in fibroblast activation and Smad transcriptional activity to promote transforming growth factor-ß (TGF-ß)-induced differentiation from fibroblasts to myofibrolasts. Macrophage stimulating 1/2 (MST1/2), a negative regulator of YAP, also increases in fibroblasts by TGF-ß stimulation. Here, we examined whether MST1/2, as a negative regulator, attenuated YAP and TGF-ß/Smad signaling in fibroblasts to reduce fibrosis. MST1/2 and YAP expression levels increased in platelet-derived growth factor receptor-α (PDGFRα)+ cells of obstructed kidneys following the increase of TGF-ß and renal fibrosis after unilateral ureteral obstruction. PDGFRα+ cell-specific knockout of Mst1/2 in mice increased unilateral ureteral obstruction-induced myofibroblast accumulation and fibrosis. In cultured fibroblasts, TGF-ß increased YAP and promoted its nucleus entry, but a high dose and prolonged treatment of TGF-ß increased the MST1/2 activation to prevent YAP from entering the nucleus. Our results indicate that MST1/2 is a negative feedback signal of TGF-ß-induced fibroblast differentiation.NEW & NOTEWORTHY Using a mouse model with macrophage stimulating 1/2 (Mst1/2) double knockout in PDGFRα+ cells and an MST1/2 inhibitor, we demonstrated that MST1/2 acted as a negative feedback signal of transforming growth factor-ß-induced fibroblast differentiation. Furthermore, we demonstrated that Hippo-MST as a negative feedback signal can decrease the renal fibrosis process. This finding contributes to our understanding of the mechanism of coregulated renal remodeling after injury.