Transcriptional Coactivator Research Articles

Role of the Hippo-YAP/TAZ Pathway in Epithelioid Hemangioendothelioma and its Potential as a Therapeutic Target.

Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular tumor arising from vascular endothelial cells. This study delves into the molecular mechanisms underlying EHE, with a specific focus on the Hippo-YAP/TAZ pathway. EHE is characterized molecularly by transcriptional co-activator with a PDZ-motif (TAZ)-calmodulin binding transcription activator 1 (CAMTA1) or Yes-associated protein (YAP)-transcription factor E3 (TFE3) fusions. YAP/TAZ, a transcription co-activator, binds to transcription factors and regulates gene expression. The YAP/TAZ and its upstream Hippo pathway are involved in cell proliferation and cell contact inhibition, regulating organ size and carcinogenesis. In addition to oncogenic effects, dysfunction or gene duplication of the Hippo pathway results in a poor prognosis due to epithelial-mesenchymal transformation of epithelial cells, stem cell transformation, and increased drug resistance. Notably, the TAZ-CAMTA1 fusion is specific to EHE, and genetic alterations in the Hippo pathway other than this fusion gene are absent in EHE. The TAZ-CAMTA1 fusion is a promising therapeutic target. This review summarizes recent advances in EHE, focusing on the role of the Hippo-YAP/TAZ pathway in EHE and its potential as a therapeutic target for drug development.

Discovery of a Highly Potent PROTAC Degrader of p300/CBP Proteins for the Treatment of Enzalutamide-Resistant Prostate Cancer.

Prostate cancer therapies against androgen receptor (AR) eventually develop lethal resistance; thus, exploring new therapeutic approaches is urgent for prostate cancer treatment. Acetyltransferase p300/CBP are key coactivators for AR-mediated transcription and represent promising therapeutic targets to inhibit AR activity in prostate cancer. We describe the design synthesis and evaluation of a new class of p300/CBP PROTAC degraders. We identified an excellent p300/CBP degrader MJP6412, which effectively induced degradation of p300/CBP proteins, downregulated AR target genes, and inhibited cell growth of human prostate cancer cell lines and enzalutamide-resistant cells with IC50 even at nanomolar concentrations. Furthermore, MJP6412 demonstrated significant inhibition of tumor growth in a VCaP xenograft model. Collectively, MJP6412 is a promising lead compound for the treatment of prostate cancer, especially enzalutamide-resistant prostate cancer.

Astragalus polysaccharide ameliorates bone marrow haematopoietic failure in mice with aplastic anaemia via the Hippo/TERT signalling pathway

ObjectiveTo study the effects of astragalus polysaccharide (APS) on aplastic anaemia (AA) in mice through modulation of the upstream and downstream effectors of the Hippo pathway. Further, we investigated the underlying mechanisms of APS in the treatment of AA to provide possible therapeutic strategies and drugs for clinical use. MethodsBALB/c mice were subjected to 4 Gy X-ray irradiation and subsequently injected with lymphocytes from DBA/2 donor mice by using the tail vein to establish an AA mouse model. Next BALB/c mice were randomly divided into five groups: control (untreated), AA (model), and three APS remedy groups - LA (low-dose APS, 200 mg/kg), MA (medium-dose APS, 400 mg/kg), and HA (high-dose APS, 800 mg/kg). The mice were sacrificed after 14 d of continuous gavage with different doses of APS or saline, and the bone marrow, spleen and liver were obtained. The haematopoietic condition, apoptosis levels, and the expression of Large Tumour Suppressor 1/2 (LATS 1/2), transcriptional co-activator of activation (TCA) Yes-associated protein (YesAP), transcriptional enhancer of adherence domain (TEAD) protein, and Telomerase Reverse Transcriptase (TERT), were analysed. ResultsCompared with control mice, mice in the AA group exhibited a reduction in peripheral blood cell counts, a significant decrease in the haematopoietic area, increased adipocyte infiltration, and overall haematopoietic failure, mirroring the clinical presentation of AA; After 14 days of treatment with different doses of APS, the APS-treated group, in comparison to the untreated AA group, showed an increase in peripheral blood cell counts (P < 0.05), an expansionof the haematopoietic area in the bone marrow, restoration of haematopoietic function (P < 0.05), and a marked reduction in adipocyte infiltration within the bone marrow(P < 0.05). Cell infiltration into the bone marrow was also reduced. Further experiments revealed that the expression levels of TERT, LATS 1/2, YAP, and TEAD were elevated in the treated mice (P < 0.05). ConclusionX-ray irradiation combined with T-lymphocyte infusion successfully established an AA mouse model, and APS administration modulated the expression of TERT and the Hippo pathway effectors LATS 1/2, YAP, and TEAD, which are implicated in the pathogenesis of AA. This modulation resulted in the restoration of bone marrow haematopoietic function in AA mice, suggesting APS as a promising therapeutic agent for the treatment of AA.

Anti-fibrogenic effect of umbilical cord-derived mesenchymal stem cell-conditioned media in human esophageal fibroblasts.

Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-β1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-β1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-β1. In TGF-β1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-β1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-β1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-β1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis

BackgroundIntestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PurposeTo investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. ResultsOur results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. ConclusionThis study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

DNAJB1-PRKACA fusion drives fibrolamellar liver cancer through impaired SIK signaling and CRTC2/p300-mediated transcriptional reprogramming.

Fibrolamellar carcinoma (FLC) is a liver cancer of adolescents and young adults characterized by fusions of the genes encoding the protein kinase A catalytic subunit, PRKACA, and heat shock protein, DNAJB1. The chimeric DNAJB1-PRKACA protein has increased kinase activity and is essential for FLC xenograft growth. Here, we explore the critical oncogenic pathways controlled by DNAJB1-PRKACA using patient-derived FLC models, engineered systems, and patient samples. We show that a core function of DNAJB1-PRKACA is the phosphorylation and inactivation of Salt-inducible kinases (SIKs). This leads to deregulation of the CRTC2 transcriptional co-activator and p300 acetyltransferase, resulting in transcriptional reprogramming and increased global histone acetylation, driving malignant growth. Our studies establish a central oncogenic mechanism of DNAJB1-PRKACA and suggest the potential of targeting CRTC2/p300 in FLC. Notably, these findings link this rare cancer's signature fusion oncoprotein to more common cancer gene alterations involving STK11 and GNAS, which also function via SIK suppression.

The G Protein-Coupled Receptor GPR56 Is an Inhibitory Checkpoint for NK Cell Migration.

G protein-coupled receptors (GPCRs) represent the largest family of surface receptors and are responsible for key physiological functions, including cell growth, neurotransmission, hormone release, and cell migration. The GPCR 56 (GPR56), encoded by ADGRG1, is an adhesion GPCR found on diverse cell types, including neural progenitor cells, melanoma cells, and lymphocytes, such as effector memory T cells, γδ T cells, and NK cells. Using RNA-sequencing and high-resolution flow cytometry, we found that GPR56 mRNA and protein expression increased with NK cell differentiation, reaching its peak in adaptive NK cells. Small interfering RNA silencing of GPR56 led to increased spontaneous and chemokine-induced migration, suggesting that GPR56 functions as an upstream checkpoint for migration of highly differentiated NK cells. Increased NK cell migration could also be induced by agonistic stimulation of GPR56 leading to rapid internalization and deactivation of the receptor. Mechanistically, GPR56 ligation and downregulation were associated with transcriptional coactivator with PDZ-binding motif translocation to the nucleus and increased actin polymerization. Together, these data provide insights into the role of GPR56 in the migratory behavior of human NK cell subsets and may open possibilities to improve NK cell infiltration into cancer tissues by releasing a migratory checkpoint.

Expression of POU2F3 Transcription Factor and POU2AF2, POU2F3 Coactivator, in Tuft Cell-like Carcinoma and Other Tumors.

Epithelial chemosensory cells in hollow organs, also known as tuft cells, were implicated in tumorigenesis, including a tuft cell-like small cell lung carcinoma. Expression of the POU2F3 transcription factor is a marker of tuft cell lineage. However, tuft cell development, differentiation, and proliferation are controlled by the expression of the complex formed by POU2F3 and POU2AF2 or POU2AF3 transcriptional coactivators. A cohort of epithelial (n=6064) and mesenchymal/neuroectodermal (n=2730) tumors was screened for POU2F3 expression by immunohistochemistry. Variable immunoreactivity ranging from diffuse to scattered positive cells was found in ∼12.4% of epithelial and 4.6% of mesenchymal/neuroectodermal tumors. Cases with predominantly diffuse or patchy POU2F3 positivity representing various types of malignant tumors (n=43) were selected for further study, including POU2AF2 immunohistochemistry. Thirteen of 15 tumors with neuroendocrine differentiation originating from the lung, colon, head and neck, skin, and bladder revealed diffuse POU2F3 positivity. Most of those tumors (n=9) co-expressed POU2AF2, usually extensively. Seven squamous and basal cell carcinomas from the oral cavity, skin, lung, and thymus with diffuse POU2F3 immunostaining except one, lacked POU2AF2 expression. Other variably POU2F3-positive carcinomas (n=13) from the colon, pancreas, liver, kidney, testis, endometrium, ovary, and breast lacked POU2AF2 immunoreactivity. All POU2F3-positive mesenchymal and neuroectodermal tumors (n=8), including synovial sarcoma, solitary fibrous tumor, glioblastoma, Wilms tumor, and melanoma were POU2AF2-negative. POU2F3 expression is a highly sensitive but nonspecific indicator of tuft cell differentiation. Co-expression of POU2F3 and POU2AF2 appears to be a more specific marker, although it may not pinpoint tumors driven by the POU2F3-POU2AF3 complex.

EDA Fibronectin Microarchitecture and YAP Translocation During Wound Closure.

Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.

Targeting CBP and p300: Emerging Anticancer Agents.

CBP and p300 are versatile transcriptional co-activators that play essential roles in regulating a wide range of signaling pathways, including Wnt/β-catenin, p53, and HIF-1α. These co-activators influence various cellular processes such as proliferation, differentiation, apoptosis, and response to hypoxia, making them pivotal in normal physiology and disease progression. The Wnt/β-catenin signaling pathway, in particular, is crucial for cellular proliferation, differentiation, tissue homeostasis, and embryogenesis. Aberrant activation of this pathway is often associated with several types of cancer, such as colorectal tumor, prostate cancer, pancreatic and hepatocellular carcinomas. In recent years, significant efforts have been directed toward identifying and developing small molecules as novel anticancer agents capable of specifically inhibiting the interaction between β-catenin and the transcriptional co-activators CBP and p300, which are required for Wnt target gene expression and are consequently involved in the regulation of tumor cell proliferation, migration, and invasion. This review summarizes the most significant and original research articles published from 2010 to date, found by means of a PubMed search, highlighting recent advancements in developing both specific and non-specific inhibitors of CBP/β-catenin and p300/β-catenin interactions. For a more comprehensive view, we have also explored the therapeutic potential of CBP/p300 bromodomain and histone acetyltransferase inhibitors in disrupting the transcriptional activation of genes involved in various signaling pathways related to cancer progression. By focusing on these therapeutic strategies, this review aims to offer a detailed overview of recent approaches in cancer treatment that selectively target CBP and p300, with particular emphasis on their roles in Wnt/β-catenin-driven oncogenesis.

P300/CBP KATs Are Critical for Maturation and Differentiation of Adult Neural Progenitors.

Epigenetic modifications play a pivotal role in the process of neurogenesis. Among these modifications, reversible acetylation fine-tunes gene expression for both embryonic and adult neurogenesis. The CBP/KAT3A and its paralogue p300/KAT3B are well-known lysine acetyltransferases with transcriptional coactivation ability that engage in neural plasticity and memory. The exclusive role of their KAT activity in neurogenesis and memory could not be addressed due to the absence of a p300/CBP modulator, which can cross the blood-brain barrier. Previous work from our laboratory has shown that a small molecule activator, TTK21, specific to CBP/p300, when conjugated to glucose-derived carbon nanospheres (CSP), is efficiently delivered to the mouse brain and could induce dendritic branching and extend long-term memory. However, the molecular mechanisms of p300 acetyltransferase activity-dependent enhanced dendritogenesis are yet to be understood. Here, we report that CSP-TTK21 treatment to primary neuronal culture derived from mouse embryo enhances the expression of five critical genes: Neurod1 (central nervous system development), Tubb3 (immature neural marker), Camk2a (synaptic plasticity and LTP), Snap25 (spine morphogenesis plasticity), and Scn2a (propagation of the action potential). Activation of these genes by inducing the p300/CBP KAT activity presumably promotes the maturation and differentiation of adult neuronal progenitors and thereby the formation of long and highly branched doublecortin-positive functional neurons in the subgranular zone of the dentate gyrus.

Brain-body mitochondrial distribution patterns lack coherence and point to tissue-specific and individualized regulatory mechanisms.

Energy transformation capacity is generally assumed to be a coherent individual trait driven by genetic and environmental factors. This predicts that some individuals should have high and others low mitochondrial oxidative phosphorylation (OxPhos) capacity across organ systems. Here, we test this assumption using multi-tissue molecular and enzymatic activities in mice and humans. Across up to 22 mouse tissues, neither mitochondrial OxPhos capacity nor mtDNA density were correlated between tissues (median r = -0.01-0.16), indicating that animals with high mitochondrial capacity in one tissue can have low capacity in other tissues. Similarly, the multi-tissue correlation structure of RNAseq-based indices of mitochondrial gene expression across 45 tissues from 948 women and men (GTEx) showed small to moderate coherence between only some tissues (regions of the same brain), but not between brain-body tissue pairs in the same person (median r = 0.01). Mitochondrial DNA copy number (mtDNAcn) also lacked coherence across organs and tissues. Mechanistically, tissue-specific differences in mitochondrial gene expression were attributable in part to i) tissue-specific activation of canonical energy sensing pathways including the transcriptional coactivator PGC-1 and the integrated stress response (ISR), and ii) proliferative activity across tissues. Finally, we identify subgroups of individuals with high mitochondrial gene expression in some tissues (e.g., heart) but low expression in others (e.g., skeletal muscle) who display different clinical phenotypic patterns. Taken together, these data raise the possibility that tissue-specific energy sensing pathways may contribute to the idiosyncratic mitochondrial distribution patterns associated with the inter-organ heterogeneity and phenotypic diversity among individuals.

Abstract C096: The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortality in U.S. men, with African American (AA) men showing higher incidence and mortality compared to European American men. In addition, AA men are more likely to be diagnosed with more aggressive PCa resulting in reduction of treatment options, especially as tumors develop therapy resistance. Thus, understanding the mechanisms underlying PCa therapy resistance is crucial for developing new therapeutic options. The upregulation of the Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) by glucocorticoid receptor (GR) is a novel mechanism of PCa therapy resistance recently identified by our group. GR upregulation in PCa bypasses the blockade of androgen receptor by inhibitors such as enzalutamide and abiraterone. LEDGF/p75 is an oncogenic transcription co-activator that promotes DNA damage repair and cancer cell survival against environmental stressors including chemotherapeutic drugs such as docetaxel (DTX). An emerging concern in PCa treatment is therapy cross-resistance, in which the molecular mechanisms of resistance to a particular drug confer resistance to another drug. Our recent studies showed that GR upregulates and interacts with LEDGF/p75 forming a transcriptional complex that includes other oncoproteins such as Menin, JPO2, c-Myc and HRP2, and promotes DTX resistance (DTX-R) in PCa cells. In addition, we reported that both GR and LEDGF/p75 are upregulated in enzalutamide resistant (ENZ-R) PCa cells. We hypothesized that the GR- LEDGF/p75 transcriptional network plays a role in PCa therapy cross-resistance to both ENZ and DTX and is a promising therapeutic target. Cell viability assays revealed that ENZ-R in PCa cells confers cross-resistance to DTX. Western blot analysis revealed upregulation of members of the GR-LEDGF/p75 network such as GR, LEDGF/p75, Menin and c-Myc in ENZ-R PCa cells, consistent with similar observations in DTX-R PCa cells. Upon knockdown of GR and LEDGF/p75 in DTX-R PCa cells, we identified by RNA-seq analysis overlapping DEGs associated with both ENZ-R and DTX-R, suggesting that the GR-LEDGF/p75 transcriptional network may contribute to therapy cross-resistance. Consistent with this, mining of publicly available RNA-seq databases identified 46 overlapping differentially expressed genes (DEGs) between ENZ-R and DTX-R PCa cells. Pathway analysis of DEGs regulated by both GR and LEDGF/p75 revealed significant enrichment of Apoptosis, p53, and DNA repair pathways, which are associated with therapy resistance. Ongoing studies are evaluating the anti-cancer properties and synergistic/additive potential of various inhibitors of the GR-LEDGF/p75 transcriptional network in cellular models of ENZ-R and DTX-R. These studies provide novel mechanistic insights into PCa therapy cross-resistance and may lead to the development of novel therapeutic strategies to reduce PCa mortality disparities. Citation Format: Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Kai Wen Cheng, Isaac Kremsky, Charles Wang, Carlos A. Casiano. The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C096.

The effects of all-trans retinoic acid on prednisolone-induced osteoporosis in zebrafish larvae

Glucocorticoids (GCs) are extensively used as anti-inflammatory and immunosuppressive medications in the long-term treatment of rheumatic disorders, respiratory diseases, renal diseases, and organ transplantation. Prolonged use of GCs can reduce bone mineral density, leading to osteoporosis (Glucocorticoid Induced Osteoporosis, GIOP) and fracture. All-trans retinoic acid (ATRA) is an active vitamin A metabolite that regulates embryonic development and adult organ function. ATRA has been found in studies to enhance osteogenesis. To examine the interventional effects of ATRA on GIOP and the mechanisms of ATRA activities, we first performed bioinformatic analysis to identify potential gene targets of ATRA. Zebrafish larvae were recruited as experimental animals, and the frequently used GC, prednisolone, was administered to larvae to construct a GIOP model. We evaluated the influence of exogenous ATRA on the activities of bone metabolic enzymes, the expression of genes linked to osteoblasts and osteoclasts, and the restoration of bone mineral density and bone mass in GIOP zebrafish larvae. Furthermore, we studied the influence of RBM14, a transcriptional coactivator and negative reciprocal factor of ATRA, on the regulation of osteoblastic gene expression during the anti-GIOP process of ATRA using the morpholino knockdown approach. The findings of bone metabolic enzyme activity (alkaline phosphatase, ALP and tartrate-resistant acid phosphatase, TRAP) and expression assays of osteoblastic marker genes (Runx2a, Runx2b, SP7, Csf1a, RANKL, and CTSK) indicated that ATRA had bidirectional effects on osteogenesis. However, in the GIOP model, ATRA reversed the GIOP-induced osteoporosis phenotype by inhibiting the GIOP-induced suppression of osteoblastic metabolic enzyme (ALP) activities and osteoblastic marker gene expression (Runx2a, Runx2b, and SP7), and this antagonism was concentration-dependent. We also observed that ATRA inhibited RBM14 expression in zebrafish larvae, while ATRA alone and RBM14 knockdown showed a consistent induction of osteoblast marker gene expression, implying that ATRA's inhibitory effect on RBM14 expression may underlie ATRA's osteogenic effects. Based on these data, we postulated that ATRA may ameliorate GIOP by decreasing RBM14 expression, thereby enhancing osteoblastic marker gene expression.

Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia

Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.

Dihydroartemisinin Inhibits Epithelial-Mesenchymal Transition Progression in Medullary Thyroid Carcinoma Through the Hippo Signaling Pathway Regulated by Interleukin-6.

Dihydroartemisinin (DHA), an artemisinin derivative, can influence certain malignancies' inflammatory response and growth. This study used Cell Counting Kit-8 and Transwell assays to show that DHA suppressed the growth, migration, and invasion of medullary thyroid cancer cells. Furthermore, the authors used enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence to confirm the expression of the transcriptional coactivators Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding domain (TAZ) downstream of the Hippo pathway and changes in the expression of the epithelial-mesenchymal transition (EMT) process markers E-cadherin and N-cadherin. These results demonstrate that DHA effectively reduced the expression of interleukin (IL)-6 in medullary thyroid carcinoma (MTC) cells and hindered the EMT process by regulating the Hippo pathway. This regulation was achieved by promoting YAP phosphorylation and inhibiting YAP/TAZ protein expression. Additional activation of the Hippo pathway by GA-017 alleviated the inhibitory effect of DHA on IL-6. Hippo pathway activation led to an increase in the expression of E-cadherin, a marker of EMT. In conclusion, DHA was demonstrated to regulate the Hippo pathway by inhibiting IL-6 secretion, leading to the inhibition of EMT in MTC. These findings provide a theoretical foundation for further exploration of the anticancer mechanisms of DHA and offer valuable insights into its potential clinical application as a combinatorial drug.

Collaboration between DELLA and MED15 to regulate transcription in plants.

Gibberellins (GA) are diterpenoids that are categorized as one of main hormones that promote major developmental responses such as germination and stem elongation in plants. DELLA proteins act as the key repressors of GA responses. They interact with hundreds of different proteins. While the functioning of DELLA as transcriptional coactivators has also been reported earlier, the actual mechanism still remains elusive. One recent report describes interaction of DELLA with Mediator subunit MED15 as one of the mechanisms contributing to its transcription activation capability (Hernández-García et al. 2024). Interestingly, this mechanism of DELLA-MED15 module-mediated transcription regulation seems to be very ancient conserved from bryophyte Marchantia polymorpha to dicot Arabidopsis thaliana.

Identifying adeno-associated virus (AAV) vectors that efficiently target high grade glioma cells, for in vitro monitoring of temporal cell responses.

To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce geneticand phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.

YAP/TAZ Signaling in the Pathobiology of Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is a severe, irreversible lung disease characterized by progressive scarring, with idiopathic pulmonary fibrosis (IPF) being the most prevalent form. IPF's pathogenesis involves repetitive lung epithelial injury leading to fibroblast activation and excessive extracellular matrix (ECM) deposition. The prognosis for IPF is poor, with limited therapeutic options like nintedanib and pirfenidone offering only modest benefits. Emerging research highlights the dysregulation of the yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathway as a critical factor in PF. YAP and TAZ, components of the Hippo pathway, play significant roles in cell proliferation, differentiation, and fibrosis by modulating gene expression through interactions with TEA domain (TEAD) transcription factors. The aberrant activation of YAP/TAZ in lung tissue promotes fibroblast activation and ECM accumulation. Targeting the YAP/TAZ pathway offers a promising therapeutic avenue. Preclinical studies have identified potential treatments, such as trigonelline, dopamine receptor D1 (DRD1) agonists, and statins, which inhibit YAP/TAZ activity and demonstrate antifibrotic effects. These findings underscore the importance of YAP/TAZ in PF pathogenesis and the potential of novel therapies aimed at this pathway, suggesting a new direction for improving IPF treatment outcomes. Further research is needed to validate these approaches and translate them into clinical practice.

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased the phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active sites were required for PTPN14 to promote differentiation. Together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.IMPORTANCEThe Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.