MAML1/2 promote YAP/TAZ nuclear localization and tumorigenesis

The Hippo pathway plays a pivotal role in tissue homeostasis and tumor suppression. YAP and TAZ are downstream effectors of the Hippo pathway, and their activities are tightly suppressed by phosphorylation-dependent cytoplasmic retention. However, the molecular mechanisms governing YAP/TAZ nuclear localization have not been fully elucidated. Here, we report that Mastermind-like 1 and 2 (MAML1/2) are indispensable for YAP/TAZ nuclear localization and transcriptional activities. Ectopic expression or depletion of MAML1/2 induces nuclear translocation or cytoplasmic retention of YAP/TAZ, respectively. Additionally, mutation of the MAML nuclear localization signal, as well as its YAP/TAZ interacting region, both abolish nuclear localization and transcriptional activity of YAP/TAZ. Importantly, we demonstrate that the level ofMAML1messenger RNA (mRNA) is regulated by microRNA-30c (miR-30c) in a cell-density-dependent manner. In vivo and clinical results suggest that MAML potentiates YAP/TAZ oncogenic function and positively correlates with YAP/TAZ activation in human cancer patients, suggesting pathological relevance in the context of cancer development. Overall, our study not only provides mechanistic insight into the regulation of YAP/TAZ subcellular localization, but it also strongly suggests that the miR30c–MAML–YAP/TAZ axis is a potential therapeutic target for developing novel cancer treatments.

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Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness

Molecular Cancer ◽

10.1186/s12943-020-1130-z ◽

2020 ◽

Vol 19 (1) ◽

Cited By ~ 7

Author(s):

Sandra Muñoz-Galván ◽

Blanca Felipe-Abrio ◽

Eva M. Verdugo-Sivianes ◽

Marco Perez ◽

Manuel P. Jiménez-García ◽

...

Keyword(s):

Ovarian Cancer ◽

Molecular Mechanisms ◽

Target Genes ◽

Hippo Pathway ◽

Ovarian Tumors ◽

Tumor Resistance ◽

Functional Link ◽

The Hippo Pathway

Abstract Background Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. Methods shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. Results We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. Conclusions Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.

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MINDY1 promotes bladder cancer progression by stabilizing YAP

Cancer Cell International ◽

10.1186/s12935-021-02095-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yongwen Luo ◽

Jun Zhou ◽

Jianing Tang ◽

Fengfang Zhou ◽

Zhiwen He ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Progression ◽

Target Genes ◽

Hippo Pathway ◽

Tumor Model ◽

Dependent Manner ◽

Interaction Domain ◽

Bona Fide ◽

The Hippo Pathway

Abstract Background Bladder cancer is one of the most commonly diagnosed urological malignant tumor. The Hippo tumor suppressor pathway is highly conserved in mammals and plays an important role in carcinogenesis. YAP is one of major key effectors of the Hippo pathway. However, the mechanism supporting abnormal YAP expression in bladder cancer remains to be characterized. Methods Western blot was used to measure the expression of MINDY1 and YAP, while the YAP target genes were measured by real-time PCR. CCK8 assay was used to detect the cell viability. The xeno-graft tumor model was used for in vivo study. Protein stability assay was used to detect YAP protein degradation. Immuno-precipitation assay was used to detect the interaction domain between MINDY1 and YAP. The ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP. Results In the present study, we identified MINDY1, a DUB enzyme in the motif interacting with ubiquitin-containing novel DUB family, as a bona fide deubiquitylase of YAP in bladder cancer. MINDY1 was shown to interact with, deubiquitylate, and stabilize YAP in a deubiquitylation activity-dependent manner. MINDY1 depletion significantly decreased bladder cancer cell proliferation. The effects induced by MINDY1 depletion could be rescued by further YAP overexpression. Depletion of MINDY1 decreased the YAP protein level and the expression of YAP/TEAD target genes in bladder cancer, including CTGF, ANKRD1 and CYR61. Conclusion In general, our findings establish a previously undocumented catalytic role for MINDY1 as a deubiquitinating enzyme of YAP and provides a possible target for the therapy of bladder cancer.

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Nerfin-1 represses transcriptional output of Hippo signaling in cell competition

eLife ◽

10.7554/elife.38843 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Pengfei Guo ◽

Chang-Hyun Lee ◽

Huiyan Lei ◽

Yonggang Zheng ◽

Katiuska Daniela Pulgar Prieto ◽

...

Keyword(s):

Dna Binding ◽

Hippo Pathway ◽

Ectopic Expression ◽

Tissue Growth ◽

Hippo Signaling ◽

Dependent Manner ◽

Cell Competition ◽

Regulatory Mode ◽

The Hippo Pathway ◽

Transcriptional Output

The Hippo tumor suppressor pathway regulates tissue growth in Drosophila by restricting the activity of the transcriptional coactivator Yorkie (Yki), which normally complexes with the TEF/TEAD family DNA-binding transcription factor Scalloped (Sd) to drive the expression of growth-promoting genes. Given its pivotal role as a central hub in mediating the transcriptional output of Hippo signaling, there is great interest in understanding the molecular regulation of the Sd-Yki complex. In this study, we identify Nerfin-1 as a transcriptional repressor that antagonizes the activity of the Sd-Yki complex by binding to the TEA DNA-binding domain of Sd. Consistent with its biochemical function, ectopic expression of Nerfin-1 results in tissue undergrowth in an Sd-dependent manner. Conversely, loss of Nerfin-1 enhances the ability of winner cells to eliminate loser cells in multiple scenarios of cell competition. We further show that INSM1, the mammalian ortholog of Nerfin-1, plays a conserved role in repressing the activity of the TEAD-YAP complex. These findings reveal a novel regulatory mode converging on the transcriptional output of the Hippo pathway that may be exploited for modulating the YAP oncoprotein in cancer and regenerative medicine.

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Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

10.1101/474379 ◽

2018 ◽

Author(s):

Jeffrey Reina ◽

Lixin Zhou ◽

Marcos R.M. Fontes ◽

Nelly Panté ◽

Nathalie Cella

Keyword(s):

Subcellular Localization ◽

Nuclear Localization ◽

Hela Cells ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Biological Activities ◽

Dependent Manner ◽

Localization Signal

AbstractMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. Anin vitronuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

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Targeting IRF3 as a YAP agonist therapy against gastric cancer

Journal of Experimental Medicine ◽

10.1084/jem.20171116 ◽

2018 ◽

Vol 215 (2) ◽

pp. 699-718 ◽

Cited By ~ 16

Author(s):

Shi Jiao ◽

Jingmin Guan ◽

Min Chen ◽

Wenjia Wang ◽

Chuanchuan Li ◽

...

Keyword(s):

Gastric Cancer ◽

Target Genes ◽

Nuclear Translocation ◽

Patient Survival ◽

Hippo Pathway ◽

Vital Role ◽

Dependent Manner ◽

Genome Wide ◽

The Hippo Pathway ◽

Innate Antiviral Immunity

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP–TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.

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Dynamic patterns of YAP1 expression and cellular localization in the developing and injured utricle

Scientific Reports ◽

10.1038/s41598-020-77775-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vikrant Borse ◽

Matthew Barton ◽

Harry Arndt ◽

Tejbeer Kaur ◽

Mark E. Warchol

Keyword(s):

Inner Ear ◽

Cellular Localization ◽

Nuclear Translocation ◽

Hippo Pathway ◽

Hippo Signaling ◽

Supporting Cells ◽

The Hippo Pathway

AbstractThe Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.

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Rox8 promotes microRNA-dependentykimessenger RNA decay

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013449117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30520-30530

Author(s):

Xiaowei Guo ◽

Yihao Sun ◽

Taha Azad ◽

H. J. Janse van Rensburg ◽

Jingjing Luo ◽

...

Keyword(s):

Messenger Rna ◽

Rna Binding ◽

Hippo Pathway ◽

Tumor Model ◽

Oncogenic Ras ◽

Collaborative Action ◽

Evolutionarily Conserved ◽

Human Ortholog ◽

The Hippo Pathway

The Hippo pathway is an evolutionarily conserved regulator of organ growth and tumorigenesis. InDrosophila, oncogenic RasV12cooperates with loss-of-cell polarity to promote Hippo pathway-dependent tumor growth. To identify additional factors that modulate this signaling, we performed a genetic screen utilizing theDrosophila RasV12/lgl−/−in vivo tumor model and identified Rox8, a RNA-binding protein (RBP), as a positive regulator of the Hippo pathway. We found thatRox8overexpression suppresses whereasRox8depletion potentiates Hippo-dependent tissue overgrowth, accompanied by altered Yki protein level and target gene expression. Mechanistically, Rox8 directly binds to a target site located in theyki3′ UTR, recruits and stabilizes the targeting of miR-8–loaded RISC, which accelerates the decay ofykimessenger RNA (mRNA). Moreover, TIAR, the human ortholog of Rox8, is able to promote the degradation ofykimRNA when introduced intoDrosophilaand destabilizesYAPmRNA in human cells. Thus, our study provides in vivo evidence that the Hippo pathway is posttranscriptionally regulated by the collaborative action of RBP and microRNA (miRNA), which may provide an approach for modulating Hippo pathway-mediated tumorigenesis.

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The p73 Tumor Suppressor Is Targeted by Pirh2 RING Finger E3 Ubiquitin Ligase for the Proteasome-dependent Degradation

Journal of Biological Chemistry ◽

10.1074/jbc.m111.261537 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35388-35395 ◽

Cited By ~ 28

Author(s):

Yong-Sam Jung ◽

Yingjuan Qian ◽

Xinbin Chen

Keyword(s):

Tumor Suppressor ◽

Ubiquitin Ligase ◽

Human Cancer ◽

Ectopic Expression ◽

E3 Ubiquitin Ligase ◽

Ring Finger ◽

Growth Suppression ◽

Dependent Manner

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.

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LPHN2 controls vascular morphogenesis by inhibiting endothelial cell adhesion and YAP/TAZ signaling

10.1101/2020.04.28.065979 ◽

2020 ◽

Author(s):

Chiara Camillo ◽

Nicola Facchinello ◽

Dafne Gays ◽

Giulia Villari ◽

Noemi Gioelli ◽

...

Keyword(s):

Endothelial Cell ◽

Molecular Mechanisms ◽

Hippo Pathway ◽

Intercellular Junctions ◽

Zebrafish Embryos ◽

Endothelial Barrier Function ◽

Vascular Morphogenesis ◽

The Hippo Pathway ◽

G Protein Coupled

SUMMARYModulation of endothelial cell (EC) adhesion to extracellular matrix (ECM) in response to mechanostimuli is essential for blood vessel patterning and functioning, yet the underpinning molecular mechanisms are deciphered only in part. We identify the adhesion G protein-coupled receptor Latrophilin 2 (LPHN2) as a novel determinant of vascular morphogenesis and endothelial barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM adhesions, signals through cAMP/Rap1, and negatively regulates ECM-elicited YAP/TAZ mechanosignaling and haptotaxis. ECs also express endogenous fibronectin-leucine-rich transmembrane 2 (FLRT2) ligand that promotes cAMP/Rap1 signaling and hinders haptotaxis via LPHN2. To validate these findings in vivo, we generated lphn2a mutant zebrafish embryos in which vascular ECs are abnormally stretched, display YAP/TAZ hyperactivation, and lack proper intercellular junctions. Consistently, intravascularly injected cancer cells extravasate more easily in lphn2a null embryos compared to control animals. Overall, we unveil in vascular ECs a novel crosstalk between LPHN2 and the Hippo pathway, that may be therapeutically exploited to interfere with cancer metastatic dissemination.

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Activated MST2 kinase is free of kinetic regulation

10.1101/2022.01.13.476221 ◽

2022 ◽

Author(s):

Thomas J Koehler ◽

Thao Tran ◽

Jennifer M Kavran

Keyword(s):

Kinetic Analysis ◽

Molecular Mechanisms ◽

Hippo Pathway ◽

Kinase Domain ◽

Caspase Cleavage ◽

Downstream Signaling ◽

Steady State Kinetic ◽

Signaling Events ◽

The Hippo Pathway

Canonically, MST1/2 functions as a core kinase of the Hippo pathway and non-canonically is both activated during apoptotic signaling and acts in concert with RASSFs in T-cells. Faithful signal transduction relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made understanding the molecular mechanisms regulating activation of MST1/2 and identifying downstream signaling events. Here we present a kinetic analysis analyzing how the ability of MST1/2 to phosphorylate substrates is regulated. Using a steady state kinetic system, we parse the contribution of different factors including the domains of MST2, phosphorylation, caspase cleavage, and complex formation to MST2 activity. In the unphosphorylated state, we find the SARAH domain stabilizes substrate binding. Phosphorylation, we also determine, drives activation of MST2 and that once activated the kinase domain is free of regulation. The binding partners SAV1, MOB1A, and RASSF5 do not alter the kinetics of phosphorylated MST2. We also show that the caspase cleaved MST2 fragment is as active as full-length suggesting that the linker region of MST2 does not inhibit the catalytic activity of the kinase domain but instead regulates MST2 activity through non-catalytic mechanisms. This kinetic analysis helps establish a framework for interpreting how signaling events, mutations, and post-translational modifications contribute to signaling of MST2 in vivo.

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