The scaffold protein disabled 2 (DAB2) and its role in tumor development and progression.

BACKGROUND: Disabled 2 (DAB2) is a multifunctional protein that has emerged as a critical component in the regulation of tumor growth. Its dysregulation is implicated in various types of cancer, underscoring its importance in understanding the molecular mechanisms underlying tumor development and progression. This review aims to unravel the intricate molecular mechanisms by which DAB2 exerts its tumor-suppressive functions within cancer signaling pathways. METHODS AND RESULTS: We conducted a comprehensive review of the literature focusing on the structure, expression, physiological functions, and tumor-suppressive roles of DAB2. We provide an overview of the structure, expression, and physiological functions of DAB2. Evidence supporting DAB2's role as a tumor suppressor is explored, highlighting its ability to inhibit cell proliferation, induce apoptosis, and modulate key signaling pathways involved in tumor suppression. The interaction between DAB2 and key oncogenes is examined, elucidating the interplay between DAB2 and oncogenic signaling pathways. We discuss the molecular mechanisms underlying DAB2-mediated tumor suppression, including its involvement in DNA damage response and repair, regulation of cell cycle progression and senescence, and modulation of epithelial-mesenchymal transition (EMT). The review explores the regulatory networks involving DAB2, covering post-translational modifications, interactions with other tumor suppressors, and integration within complex signaling networks. We also highlight the prognostic significance of DAB2 and its role in pre-clinical studies of tumor suppression. CONCLUSION: This review provides a comprehensive understanding of the molecular mechanisms by which DAB2 exerts its tumor-suppressive functions. It emphasizes the significance of DAB2 in cancer signaling pathways and its potential as a target for future therapeutic interventions.

YAP promotes the early development of temporomandibular joint bony ankylosis by regulating mesenchymal stem cell function.

To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP-with the osteogenic active zone labelled by RUNX2-tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis.

Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors ß-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and ß-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and ß-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and ß-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:ß-catenin.

Establishment of Induced Pancreatic Stem Cells by Yes-Associated Protein 1.

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.

Not too much, not too little-just the right amount: the story of YAP in the podocyte.

Chen et al. identify dysregulation of the transcriptional activator Yes-associated protein in the podocytes of diabetic mouse and human kidneys. Podocyte Yes-associated protein deficiency led to downregulation of the key transcription factor Wilms' tumor 1, and worsened podocyte injury in a mouse model of diabetic kidney injury. Yes-associated protein may therefore play a critical role in diabetic podocyte injury via regulation of Wilms' tumor 1 expression.

Regulation of TAK-TAB Complex Activation through Ubiquitylation.

Transforming growth factor-ß (TGF-ß) activated kinase 1 (TAK1), also named mitogen-activated protein kinase 7 (MAPK7), forms a pivotal signaling complex with TAK1-binding proteins (TAB1, TAB2, and TAB3), orchestrating critical biological processes, including immune responses, cell growth, apoptosis, and stress responses. Activation of TAK1 by stimuli, such as tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), and Toll-like receptors (TLRs), underscores its central role in cellular signaling. Given the critical role of the TAK1-binding protein (TAK1-TAB) complex in cellular signaling and its impact on various biological processes, this review seeks to understand how ubiquitination thoroughly regulates the TAK1-TAB complex. This understanding is vital for developing targeted therapies for diseases where this signaling pathway is dysregulated. The exploration is significant as it unveils new insights into the activity, stability, and assembly of the complex, underscoring its therapeutic potential in disease modulation.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

[Understanding New Regulatory Mechanism of TCR Signal Transduction].

Signal-transducing adaptor protein-2 (STAP-2) is a unique scaffold protein that regulates several immunological signaling pathways, including LIF/LIF receptor and LPS/TLR4 signals. STAP-2 is required for Fas/FasL-dependent T cell apoptosis and SDF-1α-induced T cell migration. Conversely, STAP-2 modulates integrin-mediated T cell adhesion, suggesting that STAP-2 is essential for several negative and positive T cell functions. However, whether STAP-2 is involved in T cell-antigen receptor (TCR)-mediated T cell activation is unknown. STAP-2 deficiency was recently reported to suppress TCR-mediated T cell activation by inhibiting LCK-mediated CD3ζ and ZAP-70 activation. Using STAP-2 deficient mice, it was demonstrated that STAP-2 is required for the pathogenesis of Propionibacterium acnes-induced granuloma formation and experimental autoimmune encephalomyelitis. Here, detailed functions of STAP-2 in TCR-mediated T cell activation, and how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases, are reviewed.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is a challenging disease, especially for patients with immunologically "cold" tumors devoid of tumor-infiltrating lymphocytes (TILs). We found that HGSC exhibits among the highest levels of MYCN expression and transcriptional signature across human cancers, which is strongly linked to diminished features of antitumor immunity. N-MYC repressed basal and induced IFN type I signaling in HGSC cell lines, leading to decreased chemokine expression and T cell chemoattraction. N-MYC inhibited the induction of IFN type I by suppressing tumor cell-intrinsic STING signaling via reduced STING oligomerization, and by blunting RIG-I-like receptor signaling through inhibition of MAVS aggregation and localization in the mitochondria. Single-cell RNA sequencing of human clinical HGSC samples revealed a strong negative association between cancer cell-intrinsic MYCN transcriptional program and type I IFN signaling. Thus, N-MYC inhibits tumor cell-intrinsic innate immune signaling in HGSC, making it a compelling target for immunotherapy of cold tumors.

Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

Dysfunctional adipocytes promote tumor progression through YAP/TAZ-dependent cancer-associated adipocyte transformation.

Obesity has emerged as a prominent risk factor for the development of malignant tumors. However, the existing literature on the role of adipocytes in the tumor microenvironment (TME) to elucidate the correlation between obesity and cancer remains insufficient. Here, we aim to investigate the formation of cancer-associated adipocytes (CAAs) and their contribution to tumor growth using mouse models harboring dysfunctional adipocytes. Specifically, we employ adipocyte-specific BECN1 KO (BaKO) mice, which exhibit lipodystrophy due to dysfunctional adipocytes. Our results reveal the activation of YAP/TAZ signaling in both CAAs and BECN1-deficient adipocytes, inducing adipocyte dedifferentiation and formation of a malignant TME. The additional deletion of YAP/TAZ from BaKO mice significantly restores the lipodystrophy and inflammatory phenotypes, leading to tumor regression. Furthermore, mice fed a high-fat diet (HFD) exhibit decreased BECN1 and increased YAP/TAZ expression in their adipose tissues. Treatment with the YAP/TAZ inhibitor, verteporfin, suppresses tumor progression in BaKO and HFD-fed mice, highlighting its efficacy against mice with metabolic dysregulation. Overall, our findings provide insights into the key mediators of CAA and their significance in developing a TME, thereby suggesting a viable approach targeting adipocyte homeostasis to suppress cancer growth.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Strengthening Molecular Glues: Design Strategies for Improving Thalidomide Analogs as Cereblon Effectors and Anticancer Agents.

In the two decades since a novel thalidomide analog was last approved, many promising drug candidates have emerged with remarkable potency as targeted protein degraders. Likewise, the advent of PROTACs for suppressing 'undruggable' protein targets reinforces the need for new analogs with improved cereblon affinity, target selectivity and drug-like properties. However, thalidomide and its approved derivatives remain plagued by several shortcomings, such as structural instability and poor solubility. Herein, we present a review of strategies for mitigating these shortcomings and highlight contemporary drug discovery approaches that have generated novel thalidomide analogs with enhanced efficacy as cereblon effectors and/or anticancer agents.

FAT1 inhibits the proliferation of DLBCL cells via increasing the m6A modification of YAP1 mRNA.

Emerging evidence shows that FAT atypical cadherin 1 (FAT1) mutations occur in lymphoma and are associated with poorer overall survival. Considering that diffuse large B cell lymphoma (DLBCL) is the category of lymphoma with the highest incidence rate, this study aims to explore the role of FAT1 in DLBCL. The findings demonstrate that FAT1 inhibits the proliferation of DLBCL cell lines by downregulating the expression of YAP1 rather than by altering its cellular localization. Mechanistic analysis via meRIP-qPCR/luciferase reporter assays showed that FAT1 increases the m6A modification of YAP1 mRNA 3'UTR and the subsequent binding of heterogeneous nuclear ribonucleoprotein D (HNRNPD) to the m6A modified YAP1 mRNA, thus decreasing the stability of YAP1 mRNA. Furthermore, FAT1 increases YAP1 mRNA 3'UTR m6A modification by decreasing the activity of the TGFß-Smad2/3 pathway and the subsequent expression of ALKBH5, which is regulated at the transcriptional level by Smad2/3. Collectively, these results reveal that FAT1 inhibits the proliferation of DLBCL cells by increasing the m6A modification of the YAP1 mRNA 3'UTR via the TGFß-Smad2/3-ALKBH5 pathway. The findings of this study therefore indicate that FAT1 exerts anti-tumor effects in DLBCL and may represent a novel target in the treatment of this form of lymphoma.

Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro.

Atrial fibrosis serves as an arrhythmogenic substrate in atrial fibrillation (AF) and contributes to AF persistence. Treating atrial fibrosis is challenging because atrial fibroblast activity is multifactorial. We hypothesized that the primary cilium regulates the profibrotic response of AF atrial fibroblasts, and explored therapeutic potentials of targeting primary cilia to treat fibrosis in AF. We included 25 patients without AF (non-AF) and 26 persistent AF patients (AF). Immunohistochemistry using a subset of the patients (non-AF: n = 10, AF: n = 10) showed less ciliated fibroblasts in AF versus non-AF. Acetylated α-tubulin protein levels were decreased in AF, while the gene expressions of AURKA and NEDD9 were highly increased in AF patients' left atrium. Loss of primary cilia in human atrial fibroblasts through IFT88 knockdown enhanced expression of ECM genes, including FN1 and COL1A1. Remarkably, restoration or elongation of primary cilia by an AURKA selective inhibitor or lithium chloride, respectively, prevented the increased expression of ECM genes induced by different profibrotic cytokines in atrial fibroblasts of AF patients. Our data reveal a novel mechanism underlying fibrotic substrate formation via primary cilia loss in AF atrial fibroblasts and suggest a therapeutic potential for abrogating atrial fibrosis by restoring primary cilia.