The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

Mutations in the enzyme isocitrate dehydrogenase 1/2 (IDH1/2) are the most common somatic mutations in low-grade glioma (LGG). The Hippo signaling pathway is known to play a key role in organ size control, and its dysregulation is involved in the development of diverse cancers. Large tumor suppressor 1/2 (LATS1/2) are core Hippo pathway components that phosphorylate and inactivate Yes-associated protein (YAP), a transcriptional co-activator that regulates expression of genes involved in tumorigenesis. A recent report from The Cancer Genome Atlas (TCGA) has highlighted a frequent hypermethylation of LATS2 in IDH-mutant LGG. However, it is unclear if LATS2 hypermethylation is associated with YAP activation and prognosis of LGG patients. Here, we performed a network analysis of the status of the Hippo pathway in IDH-mutant LGG samples and determined its association with cancer prognosis. Combining TCGA data with our biochemical assays, we found hypermethylation of LATS2 promoter in IDH-mutant LGG. LATS2 hypermethylation, however, did not translate into YAP activation but highly correlated with IDH mutation. LATS2 hypermethylation may thus serve as an alternative for IDH mutation in diagnosis and a favorable prognostic factor for LGG patients.

YAP and TAZ (YAP/TAZ), two major effectors of the Hippo signaling pathway, are frequently activated in human cancers. The activity of YAP/TAZ is strictly repressed upon phosphorylation by LATS1/2 tumor suppressors. However, it is unclear how LATS1/2 are precisely regulated by upstream factors such as Hippo kinases MST1/2. Here, we show that WWC proteins (WWC1/2/3) directly interact with LATS1/2 and SAV1, and SAV1, in turn, brings in MST1/2 to phosphorylate and activate LATS1/2. Hence, WWC1/2/3 play an organizer role in a signaling module that mediates LATS1/2 activation by MST1/2. Moreover, we have defined a minimum protein interaction interface on WWC1/2/3 that is sufficient to activate LATS1/2 in a robust and specific manner. The corresponding minigene, dubbed as SuperHippo, can effectively suppress tumorigenesis in multiple tumor models. Our study has uncovered a molecular mechanism underlying LATS1/2 regulation and provides a strategy for treating diverse malignancies related to Hippo pathway dysregulation.

Cellular senescence is a driver of various aging-associated disorders, including osteoarthritis. Here, we identified a critical role for Yes-associated protein (YAP), a major effector of Hippo signaling, in maintaining a younger state of human mesenchymal stem cells (hMSCs) and ameliorating osteoarthritis in mice. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 nuclease (Cas9)-mediated knockout (KO) of YAP in hMSCs resulted in premature cellular senescence. Mechanistically, YAP cooperated with TEA domain transcriptional factor (TEAD) to activate the expression of forkhead box D1 (FOXD1), a geroprotective protein. YAP deficiency led to the down-regulation of FOXD1. In turn, overexpression of YAP or FOXD1 rejuvenated aged hMSCs. Moreover, intra-articular administration of lentiviral vector encoding YAP or FOXD1 attenuated the development of osteoarthritis in mice. Collectively, our findings reveal YAP-FOXD1, a novel aging-associated regulatory axis, as a potential target for gene therapy to alleviate osteoarthritis.

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivation impairs tissue development and regeneration. Dynamic and precise control of YAP/TAZ activity is thus important to ensure proper physiological regulation and homeostasis of the cells. Here, we show that YAP/TAZ activation results in activation of their negative regulators, LATS1/2 (large tumor suppressor 1/2) kinases, to constitute a negative feedback loop of the Hippo pathway in both cultured cells and mouse tissues. YAP/TAZ in complex with the transcription factor TEAD (TEA domain family member) directly induce LATS2 expression. Furthermore, YAP/TAZ also stimulate the kinase activity of LATS1/2 through inducing NF2 (neurofibromin 2). This feedback regulation is responsible for the transient activation of YAP upon lysophosphatidic acid (LPA) stimulation and the inhibition of YAP-induced cell migration. Thus, this LATS-mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP/TAZ regulation.

Histones constitute a type of essential nuclear proteins important for chromatin structure and functions. The expression of major histones is strictly confined to the S phase of a cell cycle and tightly coupled to DNA replication.

Background and Aim: Biliary atresia (BA), an inflammatory destruction of the bile ducts, leads to liver fibrosis in infants and accounts for half of cases undergoing pediatric liver transplantation. Yes-associated protein (YAP), an effector of the Hippo signaling pathway, is critical in maintaining identities of bile ductal cells. Here, we evaluated the expression of YAP and YAP target genes in BA livers and a rhesus rotavirus (RRV)-induced BA mice model. Methods: Liver specimens collected from 200 BA patients were compared with those of 30 non-BA patients. Model mice liver tissues were also collected. The expression of YAP and YAP target genes were measured by transfection, RNA-seq, immunohistochemistry, immunoblot, and quantitative PCR. Masson's trichrome staining and the Biliary Atresia Research Consortium (BARC) system were utilized to score liver fibrosis status. Results: The expression of YAP is elevated and positively correlated with fibrosis in BA livers. Moreover, ANKRD1, which is identified as the target gene of YAP, is also highly expressed in BA livers. Consistent with clinical data, YAP and ANKRD1 are significantly upregulated in RRV-induced BA mouse model. Conclusions: YAP expression is closely correlated with the bile duct hyperplasia and liver fibrosis, and may serve as an indicator for liver fibrosis and BA progression. This study indicates an involvement of the Hippo signaling pathway in the development of BA, and the YAP induced ANKRD1 expression may also be related to bile duct hyperplasia in BA. This provides a new direction for more in-depth exploration of the etiology and pathogenesis of biliary atresia.

The Hippo pathway is a central regulator of organ size and tumorigenesis and is commonly depicted as a kinase cascade, with an increasing number of regulatory and adaptor proteins linked to its regulation over recent years. Here, we propose that two Hippo signaling modules, MST1/2-SAV1-WWC1-3 (HPO1) and MAP4K1-7-NF2 (HPO2), together regulate the activity of LATS1/2 kinases and YAP/TAZ transcriptional co-activators. In mouse livers, the genetic inactivation of either HPO1 or HPO2 module results in partial activation of YAP/TAZ, bile duct hyperplasia, and hepatocellular carcinoma (HCC). On the contrary, inactivation of both HPO1 and HPO2 modules results in full activation of YAP/TAZ, rapid development of intrahepatic cholangiocarcinoma (iCCA), and early lethality. Interestingly, HPO1 has a predominant role in regulating organ size. HPO1 inactivation causes a homogenous YAP/TAZ activation and cell proliferation across the whole liver, resulting in a proportional and rapid increase in liver size. Thus, this study has reconstructed the order of the Hippo signaling network and suggests that LATS1/2 and YAP/TAZ activities are finetuned by HPO1 and HPO2 modules to cause different cell fates, organ size changes, and tumorigenesis trajectories.

Proteasome inhibitors (PIs) are currently used in the clinic to treat cancers such as multiple myeloma (MM). However, cancer cells often rapidly develop drug resistance towards PIs due to a compensatory mechanism mediated by nuclear factor erythroid 2 like 1 (NFE2L1) and aspartic protease DNA damage inducible 1 homolog 2 (DDI2). Following DDI2-mediated cleavage, NFE2L1 is able to induce transcription of virtually all proteasome subunit genes. Under normal condition, cleaved NFE2L1 is constantly degraded by proteasome, whereas in the presence of PIs, it accumulates and induces proteasome synthesis which in turn promotes the development of drug resistance towards PIs. Here, we report that Nelfinavir (NFV), an HIV protease inhibitor, can inhibit DDI2 activity directly. Inhibition of DDI2 by NFV effectively blocks NFE2L1 proteolysis and potentiates cytotoxicity of PIs in cancer cells. Recent clinical evidence indicated that NFV can effectively delay the refractory period of MM patients treated with PI-based therapy. Our finding hence provides a specific molecular mechanism for combinatorial therapy using NFV and PIs for treating MM and probably additional cancers.

Natriuretic peptide receptor 2 (NPR2) plays a key role in cartilage and bone morphogenesis. The NPR2 gene mutations result in acromesomelic dysplasia, Maroteaux type (AMDM), short stature with nonspecific skeletal abnormalities (SNSK), and epiphyseal chondrodysplasia, Miura type (ECDM). However, the pathogenic mechanism remains unclear. In our study, we identified one de novo (R557C) and six novel variants (G602W, V970F, R767*, R363*, F857S, and Y306S) in five independent Chinese families with familial short stature. Three patients with heterozygous mutations (G602W, V970F, and R767*) were diagnosed with SNSK (height SD score ranged from -2.25 to -5.60), while another two with compound heterozygous mutations (R363* and F857S, R557C and Y306S) were diagnosed with AMDM (height SD score ranged from -3.10 to -5.35). Among three patients with heterozygous status, two patients before puberty initiation with rhGH treatment significantly improved their growth (height velocity 7.2 cm/year, 6.0 cm/year), and one patient in puberty had a poor response to the rhGH treatment (height velocity 2.5 cm/year). Seven NPR2 gene variants were constructed and overexpressed in HEK293T and ATDC5 cells, and we found that ATDC5 cells with mutant NPR2 gene showed decreased differentiation, as evidenced by lower expression of ColII, ColX, and BMP4 and higher expression of Sox9. Moreover, the apoptosis rate was elevated in ATDC5 cells expressing the mutant NPR2 gene. N-glycosylation modification, plasma membrane localization, and ER stress resulted from the accumulation of mutant protein in ER, as shown by the higher expression of GRP78 and p-IRE1α. Overall, our results provide a novel insight into NPR2 loss of function, which could promote chondrocyte apoptosis and repress cell differentiation through ER stress and the unfolded protein response.

WWC1 regulates episodic learning and memory, and genetic nucleotide polymorphism of WWC1 is associated with neurodegenerative diseases such as Alzheimer's disease. However, the molecular mechanism through which WWC1 regulates neuronal function has not been fully elucidated. Here, we show that WWC1 and its paralogs (WWC2/3) bind directly to angiomotin (AMOT) family proteins (Motins), and recruit USP9X to deubiquitinate and stabilize Motins. Deletion of WWC genes in different cell types leads to reduced protein levels of Motins. In mice, neuron-specific deletion of Wwc1 and Wwc2 results in reduced expression of Motins and lower density of dendritic spines in the cortex and hippocampus, in association with impaired cognitive functions such as memory and learning. Interestingly, ectopic expression of AMOT partially rescues the neuronal phenotypes associated with Wwc1/2 deletion. Thus, WWC proteins modulate spinogenesis and cognition, at least in part, by regulating the protein stability of Motins.

TERT reactivation occurs frequently in human malignancies, especially advanced cancers. However, in vivo functions of TERT reactivation in cancer progression and the underlying mechanism are not fully understood. In this study, we expressed TERT and/or active BRAF (BRAF V600E) specifically in mouse thyroid epithelium. While BRAF V600E alone induced papillary thyroid cancer (PTC), coexpression of BRAF V600E and TERT resulted in poorly differentiated thyroid carcinoma (PDTC). Spatial transcriptome analysis revealed that tumors from mice coexpressing BRAF V600E and TERT were highly heterogeneous, and cell dedifferentiation was positively correlated with ribosomal biogenesis. Mechanistically, TERT boosted ribosomal RNA (rRNA) expression and protein synthesis by interacting with multiple proteins involved in ribosomal biogenesis. Furthermore, we found that CX-5461, an rRNA transcription inhibitor, effectively blocked proliferation and induced redifferentiation of thyroid cancer. Thus, TERT promotes thyroid cancer progression by inducing cancer cell dedifferentiation, and ribosome inhibition represents a potential strategy to treat TERT-reactivated cancers.

The Hippo tumor-suppressor pathway is frequently dysregulated in human cancers and represents a therapeutic target. However, strategies targeting the mammalian Hippo pathway are limited because of the lack of a well-established cell-surface regulator. Here, we show that transmembrane protein KIRREL1, by interacting with both SAV1 and LATS1/2, promotes LATS1/2 activation by MST1/2 (Hippo kinases), and LATS1/2 activation, in turn, inhibits activity of YAP/TAZ oncoproteins. Conversely, YAP/TAZ directly induce the expression of KIRREL1 in a TEAD1-4-dependent manner. Indeed, KIRREL1 expression positively correlates with canonical YAP/TAZ target gene expression in clinical tumor specimens and predicts poor prognosis. Moreover, transgenic expression of KIRREL1 effectively blocks tumorigenesis in a mouse intrahepatic cholangiocarcinoma model, indicating a tumor-suppressor role of KIRREL1. Hence, KIRREL1 constitutes a negative feedback mechanism regulating the Hippo pathway and serves as a cell-surface marker and potential drug target in cancers with YAP/TAZ dependency.

The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members--Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7-as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway.

Mammalian cells are surrounded by neighbouring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes1. The Hippo pathway effectors YAP (also known as YAP1) and TAZ (also known as WWTR1) are regulated by mechanical cues and mediate cellular responses to ECM stiffness2,3. Here we identified the Ras-related GTPase RAP2 as a key intracellular signal transducer that relays ECM rigidity signals to control mechanosensitive cellular activities through YAP and TAZ. RAP2 is activated by low ECM stiffness, and deletion of RAP2 blocks the regulation of YAP and TAZ by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, which activates RAP2 through PDZGEF1 and PDZGEF2 (also known as RAPGEF2 and RAPGEF6). At low stiffness, active RAP2 binds to and stimulates MAP4K4, MAP4K6, MAP4K7 and ARHGAP29, resulting in activation of LATS1 and LATS2 and inhibition of YAP and TAZ. RAP2, YAP and TAZ have pivotal roles in mechanoregulated transcription, as deletion of YAP and TAZ abolishes the ECM stiffness-responsive transcriptome. Our findings show that RAP2 is a molecular switch in mechanotransduction, thereby defining a mechanosignalling pathway from ECM stiffness to the nucleus.

Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in regulation of cell cycle events and cellular metabolism. The expression of Txnip is known to be induced by glucose, adenosine-containing molecules, and other physiological cues; however, the underlying regulatory mechanisms remain elusive.

Yes-associated protein (YAP) is a key downstream effector of the highly conserved Hippo signaling pathway, which regulates organ size, regeneration and tumorigenesis. Known classically to function as a transcriptional co-activator, YAP interacts with TEA domain transcription factors (TEAD1-4) to induce expression of target genes. However, a number of genes are repressed upon YAP activation, suggesting a transcriptional repressor role of YAP. Here, we report that TP73 is a direct target gene of YAP, and its transcription is repressed by YAP in a TEAD-independent manner. On the other hand, WW domains of YAP are indispensable for the regulation of TP73 expression, which may recruit YAP to TP73 gene though interaction with ZEB1 and/or RUNX2, two transcriptional repressors. Moreover, YAP-mediated repression of TP73 promotes cancer cell survival in the presence of chemotherapeutic agents, suggesting YAP-TP73 signaling as a mechanism for cancer cell resistance to chemotherapies.

Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.