LATS Kinases Research Articles

Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases—the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

LATS1 Is a Mediator of Melanogenesis in Response to Oxidative Stress and Regulator of Melanoma Growth.

The LATS1 kinase has been described as a tumor suppressor in various cancers. However, its role in melanoma has not been fully elucidated. There are several processes involved in tumorigenesis, including melanin production. Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell. Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth. We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response. We showed that LATS1 ablation significantly decreased the melanogenesis markers’ expression and melanin synthesis in melanocyte and melanoma cell lines. Moreover, silencing LATS1 resulted in enhanced oxidative stress. Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin’s protective role in this process. The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth. Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.

TRIP6 is required for tension at adherens junctions.

Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

WBP2 promotes gastric cancer cell migration via novel targeting of LATS2 kinase in the Hippo tumor suppressor pathway.

Dysregulation of signaling pathways is responsible for many human diseases. The lack of understanding of the molecular etiology of gastric cancer (GC) poses a substantial challenge to the development of effective cancer therapy. To better understand the molecular mechanisms underlying the pathogenesis of GC, which will facilitate the identification and development of effective therapeutic approaches to improve patient outcomes, mass spectrometry-based phosphoproteomics analysis was performed to map the global molecular changes in GC. A total of 530 proteins with altered phosphorylation levels were detected across a panel of 15 normal and GC cell lines. WW domain-binding protein 2 (WBP2) was validated to be upregulated in a subset of GC cell lines. WBP2 is overexpressed in 61% cases of GC compared to non-cancer tissues and high WBP2 expression correlates with poor clinical outcomes. WBP2 was found to be required for GC cell migration but is dispensable for cell growth and proliferation. WBP2 knockdown increased p-LATS2 with a concomitant increase in p-YAP, resulting in the cytoplasmic retention of YAP and ultimately the inhibition of YAP/TEAD activity and downregulation of TEAD target genes--CTGF and CYR61. Importantly, the loss of LATS2 reversed the activation of Hippo pathway caused by WBP2 knockdown, indicating that WBP2 acts through LATS2 to exert its function on the Hippo pathway. Moreover, WBP2 interacted with LATS2 to inhibit its phosphorylation and activity. In conclusion, our study established a pivotal role for WBP2 in the promotion of GC cell migration via a novel mechanism that inactivates the Hippo pathway transducer LATS2.

MYBL2 disrupts the Hippo-YAP pathway and confers castration resistance and metastatic potential in prostate cancer.

Rationale: Resistance to androgen-deprivation therapy (ADT) associated with metastatic progression remains a challenging clinical task in prostate cancer (PCa) treatment. Current targeted therapies for castration-resistant prostate cancer (CRPC) are not durable. The exact molecular mechanisms mediating resistance to castration therapy that lead to CRPC progression remain obscure.Methods: The expression of MYB proto-oncogene like 2 (MYBL2) was evaluated in PCa samples. The effect of MYBL2 on the response to ADT was determined by in vitro and in vivo experiments. The survival of patients with PCa was analyzed using clinical specimens (n = 132) and data from The Cancer Genome Atlas (n = 450). The mechanistic model of MYBL2 in regulating gene expression was further detected by subcellular fractionation, western blotting, quantitative real-time PCR, chromatin immunoprecipitation, and luciferase reporter assays.Results: MYBL2 expression was significantly upregulated in CRPC tissues and cell lines. Overexpression of MYBL2 could facilitate castration-resistant growth and metastatic capacity in androgen-dependent PCa cells by promoting YAP1 transcriptional activity via modulating the activity of the Rho GTPases RhoA and LATS1 kinase. Importantly, targeting MYBL2, or treatment with either the YAP/TAZ inhibitor Verteporfin or the RhoA inhibitor Simvastatin, reversed the resistance to ADT and blocked bone metastasis in CRPC cells. Finally, high MYBL2 levels were positively associated with TNM stage, total PSA level, and Gleason score and predicted a higher risk of metastatic relapse and poor prognosis in patients with PCa.Conclusions: Our results reveal a novel molecular mechanism conferring resistance to ADT and provide a strong rationale for potential therapeutic strategies against CRPC.

Accurate prediction of kinase-substrate networks using knowledge graphs.

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).

Heat stress activates YAP/TAZ to induce the heat shock transcriptome.

The Hippo pathway plays critical roles in cell growth, differentiation, organ development, and tissue homeostasis whereas its dysregulation can lead to tumorigenesis. YAP/TAZ are transcription co-activators and represent the main downstream effectors of the Hippo pathway. Here we show that heat stress induces a strong and rapid YAP dephosphorylation and activation. The effect of heat shock on YAP is dominant to other signals known to modulate the Hippo pathway. Heat shock inhibits LATS kinase by promoting HSP90-dependent LATS interaction with and inactivation by PP5 phosphatase. Heat shock also induces LATS ubiquitination and degradation. YAP/TAZ are crucial for cellular heat shock responses, including the heat shock transcriptome and cell viability. This study uncovers previously unknown mechanisms of Hippo regulation by heat shock as well as physiological functions of YAP in heat stress response. Our observations also reveal a potential combinational therapy involving hyperthermia and targeting of the Hippo pathway.

T851I mutation of human large tumor suppressor 1 disrupts its kinase activity and tumor-suppressor functions

AimLarge tumor suppressor 1 (LATS1) is a Ser/Thr kinase to mediate Hippo signaling pathway and plays a pivotal role in tumor suppression. By searching the COSMIC database, we found a somatic missense mutation (NM_004690.4:c.2552C>T) of human LATS1 (NP_004681.1:p.851T>I) in two colorectal cancer cell lines, and investigated the role and underlying mechanism of this mutation in the colorectal tumorigenesis. Main methodsWe performed structural and biochemistry analyses to investigate the role of LATS1 T851I mutation in Hippo signaling activation and used the mouse xenograft model to assess the role of this mutation in the colorectal tumorigenesis. Key findingsBy structural and biochemistry approaches, we propose that T851 is an active residue other than Ser909 on the activation loop and is essential for LATS1 phosphorylation and kinase activity. We then reveal that T851I mutation in LATS1 not only destabilizes the phospho-Thr1079-LATS1, a prerequisite of LATS1 kinase activity, but also reduces its binding to the downstream effectors, YAP and TAZ. As a result, T851I mutation in LATS1 attenuates Hippo signaling and decreases its tumor-suppressor functions in the colorectal cancer. SignificanceThe present study identifies the T851 as an essential residue for LATS1 kinase activity and uncovers the T851I mutation of LATS1 and consequent Hippo signaling suppression as a hitherto uncharacterized mechanism controlling colorectal tumorigenesis.

Generation of a serine/threonine-protein kinase LATS1 gene-edited iPSC MUSIi012-A-3.

In mammals, there are a number of kinases, including serine/threonine-protein kinase LATS1, that act as a core kinase of the Hippo pathway and that negatively regulate the Hippo effector protein YAP and its paralog TAZ. Using CRISPR/Cas9 technology, we established a stable LATS1 knockdown (LATS1-KD) iPSC from the MUSIi012-A cell line. The LATS1-KD iPSC MUSIi012-A-3 that was developed maintained both the normal karyotype and the pluripotent phenotype, and retained the ability to differentiate into all three embryonic germ layers.

Abstract A01: YAP1 drives ependymoma-like tumor formation in the brain

Abstract Ependymoma is the third most common pediatric brain cancer. YAP1 gene fusions have been observed in a subset of pediatric ependymomas. Here we show that increased YAP1 activity has a causative role in ependymoma-like tumor formation. YAP1 protein is strongly expressed and localized in the nucleus in a subset of human ependymomas. In the developing brain, YAP1 expression is normally found in subventricular zone neural progenitor cells, and NEX/NeuroD6-Cre induced conditional expression of active nuclear YAP1 (YAP1-5SAnls) is sufficient to drive brain tumor formation. YAP1 drives tumor formation by maintaining a nestin positive neural stem cell-like specification and preventing hippocampal neuronal differentiation. Dual conditional deletion of LATS1 and LATS2 kinases in the NEX-Cre lineage also generates similar tumors. Genetic rescue experiments in mice show that either YAP1 or its homolog TAZ can drive tumor formation downstream of LATS1/2 kinases. YAP1/TAZ activity causes tumors that display histologic and molecular characteristics of ependymoma, including ultrastructural features microvilli and tight junctions. Our results show that disruption of YAP1/TAZ activity in neuronal precursor cells leads to ependymoma-like tumors. Citation Format: Noreen Eder, Marie-Charlotte Dolmart, Suzanne Claxton, Jennifer Cotton, Jun-Hao Mao, Bram Snijders, Federico Roncaroli, Barry Thompson, Sila Ultanir. YAP1 drives ependymoma-like tumor formation in the brain [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A01.

Abstract B06: Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway

Abstract We performed a large-scale profiling of currently available kinase inhibitors (KI) to identify “off-targets” that could be repurposed/repositioned as the starting point for the rational development of novel and selective kinase inhibitors against underdrugged targets. We profiled 257 KI at a single dose against a panel of 365 wild-type kinases tested individually, for a total of about 100,000 in vitro drug/kinase data points. After unsupervised hierarchical clustering of this dataset based on percent inhibition values, we identified a well-resolved subcluster driven by 5 KI with limited polypharmacology and a clean kinase-inhibition profile, mostly targeting AGC group kinases. These inhibitors were the AKT inhibitors GSK-690693, AZD-5363, and AT-7867; the MSK inhibitor SB-747651-A; and the Rho kinases (ROCK) inhibitor GSK-269962A. Notably, the AGC and hippo kinases LATS1 and LATS2 were part of this subcluster and were both inhibited to significant extents by these 5 compounds. With the regenerative potential of a candidate LATS1/2 inhibitor in mind, we next developed several orthogonal biochemical and cellular assays to prioritize compounds that possessed the desired activity against LATS1/2 without the burden of cytotoxic “off-targets,” such as those resulting from strong AKT inhibition. Based on the results of these assays, we prioritized one of the initially identified kinase inhibitors as the starting point for an ongoing, rational structure-activity relationship (SAR) effort to develop novel, 1st-in-class, highly selective LATS1 and LATS2 inhibitors. We will present data showing how such novel LATS1/2 inhibitors efficiently block LATS1/2-dependent YAP phosphorylation, induce YAP nuclear translocation in a high cell-density setting, and promote the transcription of bona fide YAP target genes such as AMOTL2, CTGF, and CYR61. Finally, consistently with the activation of a YAP-dependent transcriptional program, small-molecule inhibitors of LATS1/2 kinases promoted proliferation/regeneration in an in vitro wound-healing assay. Altogether, we identified novel small molecules that can be used as chemical probes to acutely inactivate the Hippo signaling pathway and validated LATS1 and LATS2 kinases as novel druggable kinases whose inhibition could be exploited for several indications, spanning from regenerative medicine to cancer immunotherapy. Citation Format: Michele Ceribelli, Patrick Morris, Damien Duveau, Frances A. Tosto, Scott Hoyt, Craig J. Thomas. Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B06.

Abstract IA11: Hippo signaling Is Intrinsically regulated during cell cycle progression

Abstract The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration, and its dysregulation is widely implicated in various cancers. Hippo signaling has been recently identified as a major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival, and hepatocellular carcinoma (HCC) formation. We have found that Hippo/YAP signaling cell-autonomously inhibits HCC proliferation by interacting with Wnt and Notch signaling. We have also found that Hippo/YAP signaling cell-nonautonomously inhibits HCC by shaping its inflammatory microenvironment. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, LATS kinases and YAP/TAZ transcription factors, are regulated during cell cycle progression by core cell cycle machinery components. Furthermore, we show in Drosophila eye and wing development that cell cycle is required in vivo to regulate Hippo signaling with a conserved mechanism. Cell cycle control of Hippo signaling represents a previously unappreciated and evolutionarily conserved new layer of Hippo signaling regulation. Citation Format: Yingzi Yang, Wantae Kim, Xiaohui Wang. Hippo signaling Is Intrinsically regulated during cell cycle progression [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA11.

Abstract B07: NUAK2 inhibition for prostate cancer

Abstract Obesity is a US and global epidemic that exacerbates various cancers including prostate cancer (PC). A recent meta-analysis of men with PC found a 20% increased risk of PC-specific mortality for each 5 kg/m2 increase in BMI. To identify ways to exploit this, we performed an in vivo screen to identify kinases that were causally involved in tumor/growth in obese mice. This resulted in the identification of the AMPK-related kinase SNARK a.k.a NUAK2, a YAP1 target gene and positive feedback regulator by inhibition LATS1 kinase. To identify obesity essential kinases LAPC-4 cells were inoculated with an shRNA library of ~5,000 lentivirus targeting 513 kinases. 5 × 106 cells were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. NAUK2 shRNAs (3 of 10) were significantly depleted in LAPC4 xenograft tumors from mice on a Western diet (WD), but not tumors from mice on an isocaloric 25% calorie-restricted (CR) diet. Analysis of NUAK2 gene expression in human PCs shows NUAK2 expression is higher in PC tissue vs. normal benign. In addition, Cox regression analysis showed that NUAK2 is associated with a 3-fold increased chance of developing metastasis disease in high NUAK2 group. Proteomic data from metastatic PC rapid autopsy biopsies shows NUAK2 phospho-target LATS1 Ser464 is higher in metastatic PCs vs. benign localized prostatic tissues. Western blot analysis of PC cell lines shows NUAK2 is elevated in comparison to primary prostate epithelial cells. To test if NUAK2 is an actionable target in PC cells, we utilized two narrow-spectrum kinase inhibitors from the literature, WZ4003 and HTH-02-006. Pharmacologic inhibition of NUAK2 slows 2D PC cell growth and 3D PC spheroid growth of various PC cell lines with an IC50 range between 1-10 uM, with HTH-02-006 being the more potent. In addition, NUAK2 kinase inhibition with WZ4003 and HTH-02-006 significantly slowed migration and Matrigel invasion of PC3 and DU145 cells. To test if NUAK2 is associated with obesity-enhanced PC progression, we tested NUAK2 inhibition in combination with palmitic acid supplementation. Our preliminary studies show that pharmacologic inhibition of NUAK2 sensitized PC cells to fatty acid-induced cell death. In conclusion, our in vivo shRNA screen identified NUAK2 as an essential kinase for tumors grown in obese mice. NUAK2 expression correlates with worse outcomes in PC and NUAK2 kinase activity is elevated in metastatic vs. localized PCs. Our preliminary data show that pharmacologic inhibition of NUAK2 slows PC cell growth and migration/invasion. Lastly, our preliminary studies show that NUAK2 may lower the threshold for fatty acid-induced toxicity. Together, these preliminary data suggest NUAK2 is a relevant actionable target in PC and may synergize with obese host physiology to slow PC tumor growth. Citation Format: Weiwei Fu, Megan Zhao, Amelia Schirmer, Erick J. Maravilla, Junhee Yoon, Sungyong You, Stephen J. Freedland, Everardo Macias. NUAK2 inhibition for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B07.

Abstract B24: Identification of YAP modulators using genome-wide gain-of-function screening

Abstract The Hippo pathway is a signaling pathway that is crucial to regulate tissue homeostasis and organ growth. The core of the Hippo pathway consists of the MST1/2 and LATS1/2 kinase cascade that regulates the activity of the transcriptional coactivators YAP/TAZ by phosphorylation. Unphosphorylated YAP/TAZ display nuclear localization and interact with TEAD proteins to regulate gene expression. A tight regulation is crucial, since deregulated YAP/TAZ can lead to several organismal defects including cancer development. To ensure robustness of the signaling pathway to perturbations, many Hippo components are organized in a redundant manner, e.g., redundancy of LATS kinases. This feature aggravates the identification of new Hippo signaling components using loss-of-function screens since a knockout of one factor could potentially be compensated for by its redundant counterpart. To discover novel potential regulators of YAP, we performed a genome-scale gain-of-function CRISPR/Cas9 screening in MCF10A cells using the synergistic activation mediator (SAM) system. SAM is an adaptation of CRISPR/Cas9 to overexpress endogenous genes by targeting the proximal promoter with sgRNAs rather than knocking out target genes. Using the nuclear localization of YAP as a measure of YAP activity, we could identify several genes as positive regulators of YAP. One of the best screening hits is ISOC1 (Isochorismatase domain-containing protein 1). We validated the screening by using single sgRNAs to induce the endogenous ISOC1 locus by SAM. The expression of YAP-target genes as ANKRD1, CTGF, and IGFBP3 was increased upon ISOC1 overexpression. Main goals in the future will be to understand the biochemical mechanism by which ISOC1 modulates YAP activity and the biologic role of ISOC1. Since ISOC1 is reported to localize to peroxisomes and is induced upon dietary restriction in mice, we hypothesize that ISOC1 acts as a central hub to connect peroxisomal metabolism and YAP/TAZ activity. Citation Format: Paul Cramer, Björn von Eyss. Identification of YAP modulators using genome-wide gain-of-function screening [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B24.

P190A RhoGAP induces CDH1 expression and cooperates with E-cadherin to activate LATS kinases and suppress tumor cell growth

The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.

SASH1 suppresses triple-negative breast cancer cell invasion through YAP-ARHGAP42-actin axis.

Triple-negative breast cancer (TNBC) is extremely aggressive and lacks effective therapy. SAM and SH3 domain containing1 (SASH1) has been implicated in TNBC as a candidate tumor suppressor; however, the mechanisms of action of SASH1 in TNBC remain underexplored. Here, we show that SASH1 was significantly downregulated in TNBC patients samples compared with other subtypes of breast cancer. Ectopic SASH1 expression inhibited, while depletion of SASH1 enhanced, the invasive phenotype of TNBC cells, accompanied by deregulated expression of MMP2 and MMP9. The functional effects of SASH1 depletion were confirmed in the chicken chorioallantoic membrane and mouse xenograft models. Mechanistically, SASH1 knockdown downregulated the phosphorylation levels of the Hippo kinase LATS1 and its effector YAP (Yes associated protein), thereby upregulating YAP accumulation together with its downstream target CYR61. Consistently, forced SASH1 expression exhibited opposite effects. Pharmacological inhibition of YAP or knockdown of YAP reversed the enhanced cell invasion of TNBC cells following SASH1 depletion. Furthermore, SASH1-induced YAP signaling was LATS1-dependent, which in reverse enhanced phosphorylation of SASH1. The SASH1 S407A mutant (phosphorylation deficient) failed to rescue the altered YAP signaling by SASH1 knockdown. Notably, SASH1 depletion upregulated ARHGAP42 levels via YAP-TEAD and the YAP-ARHGAP42-actin axis contributed to SASH1-regulated TNBC cell invasion. Therefore, our findings uncover a new mechanism for the tumor-suppressive activity of SASH1 in TNBC, which may serve as a novel target for therapeutic intervention.

O-GlcNAcylation on LATS2 disrupts the Hippo pathway by inhibiting its activity

The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.

YAP1/TAZ drives ependymoma-like tumour formation in mice

YAP1 gene fusions have been observed in a subset of paediatric ependymomas. Here we show that, ectopic expression of active nuclear YAP1 (nlsYAP5SA) in ventricular zone neural progenitor cells using conditionally-induced NEX/NeuroD6-Cre is sufficient to drive brain tumour formation in mice. Neuronal differentiation is inhibited in the hippocampus. Deletion of YAP1’s negative regulators LATS1 and LATS2 kinases in NEX-Cre lineage in double conditional knockout mice also generates similar tumours, which are rescued by deletion of YAP1 and its paralog TAZ. YAP1/TAZ-induced mouse tumours display molecular and ultrastructural characteristics of human ependymoma. RNA sequencing and quantitative proteomics of mouse tumours demonstrate similarities to YAP1-fusion induced supratentorial ependymoma. Finally, we find that transcriptional cofactor HOPX is upregulated in mouse models and in human YAP1-fusion induced ependymoma, supporting their similarity. Our results show that uncontrolled YAP1/TAZ activity in neuronal precursor cells leads to ependymoma-like tumours in mice.

YAP Mediates Hair Cell Regeneration in Balance Organs of Chickens, But LATS Kinases Suppress Its Activity in Mice.

Loss of sensory hair cells causes permanent hearing and balance deficits in humans and other mammals, but for nonmammals such deficits are temporary. Nonmammals recover hearing and balance sensitivity after supporting cells proliferate and differentiate into replacement hair cells. Evidence of mechanical differences between those sensory epithelia and their supporting cells prompted us to investigate whether the capacity to activate YAP, an effector in the mechanosensitive Hippo pathway, correlates with regenerative capacity in acceleration-sensing utricles of chickens and mice of both sexes. After hair cell ablation, YAP accumulated in supporting cell nuclei in chicken utricles and promoted regenerative proliferation, but YAP remained cytoplasmic and little proliferation occurred in mouse utricles. YAP localization in supporting cells was also more sensitive to shape change and inhibition of MST1/2 in chicken utricles than in mouse utricles. Genetic manipulations showed that in vivo expression of the YAP-S127A variant caused robust proliferation of neonatal mouse supporting cells, which produced progeny that expressed hair cell markers, but proliferative responses declined postnatally. Expression of YAP-5SA, which more effectively evades inhibitory phosphorylation, resulted in TEAD-dependent proliferation of striolar supporting cells, even in adult utricles. Conditional deletion of LATS1/2 kinases abolished the inhibitory phosphorylation of endogenous YAP and led to striolar proliferation in adult mouse utricles. The findings suggest that damage overcomes inhibitory Hippo signaling and facilitates regenerative proliferation in nonmammalian utricles, whereas constitutive LATS1/2 kinase activity suppresses YAP-TEAD signaling in mammalian utricles and contributes to maintaining the proliferative quiescence that appears to underlie the permanence of sensory deficits.SIGNIFICANCE STATEMENT Loud sounds, ototoxic drugs, infections, and aging kill sensory hair cells in the ear, causing irreversible hearing loss and balance deficits for millions. In nonmammals, damage evokes shape changes in supporting cells, which can divide and regenerate hair cells. Such shape changes are limited in mammalian ears, where supporting cells develop E-cadherin-rich apical junctions reinforced by robust F-actin bands, and the cells fail to divide. Here, we find that damage readily activates YAP in supporting cells within balance epithelia of chickens, but not mice. Deleting LATS kinases or expressing YAP variants that evade LATS-mediated inhibitory phosphorylation induces proliferation in supporting cells of adult mice. YAP signaling eventually may be harnessed to overcome proliferative quiescence that limits regeneration in mammalian ears.

Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface.

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.