Transcriptional Co-activator Activity Research Articles

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 “K4me3-K9me3” bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

Screening and identification of key chromatin regulator biomarkers for ankylosing spondylitis and drug prediction: evidence from bioinformatics analysis

BackgroundAnkylosing spondylitis (AS) is one of the most common immune-mediated arthritic diseases worldwide. Despite considerable efforts to elucidate its pathogenesis, the molecular mechanisms underlying AS are still not fully understood.MethodsTo identify candidate genes involved in AS progression, the researchers downloaded the microarray dataset GSE25101 from the Gene Expression Omnibus (GEO) database. They identified differentially expressed genes (DEGs) and functionally enriched them for analysis. They also constructed a protein–protein interaction network (PPI) using STRING and performed cytoHubba modular analysis, immune cell and immune function analysis, functional analysis and drug prediction.The results showed that DEGs were mainly associated with histone modifications, chromatin organisation, transcriptional coregulator activity, transcriptional co-activator activity, histone acetyltransferase complexes and protein acetyltransferase complexes.ResultsThe researchers analysed the differences in expression between the CONTROL and TREAT groups in terms of immunity to determine their effect on TNF-α secretion. By obtaining hub genes, they predicted two therapeutic agents, AY 11–7082 and myricetin.ConclusionThe DEGs, hub genes and predicted drugs identified in this study contribute to our understanding of the molecular mechanisms underlying the onset and progression of AS. They also provide candidate targets for the diagnosis and treatment of AS.

Nuclear condensates of YAP fusion proteins alter transcription to drive ependymoma tumourigenesis.

Nuclear localization of HIPPO-YAP fusion proteins has been implicated in supratentorial ependymoma development. Here, unexpectedly, we find that liquid-liquid phase separation, rather than nuclear localization, of recurrent patient-derived YAP fusions, YAP-MAMLD1 and C11ORF95-YAP, underlies ependymoma tumourigenesis from neural progenitor cells. Mutagenesis and chimaera assays demonstrate that an intrinsically disordered region promotes oligomerization of the YAP fusions into nuclear, puncta-like, membrane-less condensates. Oligomerization and nuclear condensates induced by YAP fusion with a coiled-coil domain of transcriptional activator GCN4 also promote ependymoma formation. YAP-MAMLD1 concentrates transcription factors and co-activators, including BRD4, MED1 and TEAD, in condensates while excluding transcriptional repressive PRC2, and induces long-range enhancer-promoter interactions that promote transcription and oncogenic programmes. Blocking condensate-mediated transcriptional co-activator activity inhibits tumourigenesis, indicating a critical role of liquid phase separation for YAP fusion oncogenic activity in ependymoma. YAP fusions containing the intrinsically disordered region features are common in human tumours, suggesting that nuclear condensates could be targeted to treat YAP-fusion-induced cancers.

HSP90β chaperoning SMURF1-mediated LATS proteasomal degradation in the regulation of bone formation

Heat shock protein 90 (HSP90) molecular chaperone is responsible for the stabilization and biological activity of a diverse set of client proteins. We have previously demonstrated that inhibition of HSP90 by 17-Demethoxy-17-allyaminogeldanmycin (17-AAG) not only reverses the glucocorticoid-induced bone loss but also enhances the basal level of bone mass in mice. Here, we investigate the potential mechanism underlying HSP90-associated osteoblast differentiation and bone formation. Knockdown of HSP90β but not HSP90α or inhibition of HSP90 by 17-AAG or NVP-BEP800 negates the protein levels of large tumor suppressor (LATS), the core kinases of Hippo signaling, resulting in the inactivation of LATS and activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), in the enhancement of osteoblastic differentiation. In contrast, genetic ablation of Lats1 in mesenchymal stem cells is sufficient to abolish the HSP90 inhibition-induced osteoblastic differentiation and bone formation. Mechanistically, HSP90β but not HSP90α chaperones and prevents the SMAD specific E3 ubiquitin protein ligase 1 (SMURF1)-mediated and ubiquitination-dependent LATS protein proteasomal degradation, whereas 17-AAG abolishes these effects of HSP90β. Thus, these results uncover the HSP90β chaperoning SMURF1-mediated LATS protein proteasomal degradation and the subsequent YAP/TAZ activation as a hitherto uncharacterized mechanism controlling osteoblastic differentiation and bone formation.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

SIRT6 Protects Against Liver Fibrosis by Negatively Regulating YAP/TAZ

Hepatic fibrosis often occurs after chronic liver injury, and it is largely attributable to the activation of hepatic stellate cells (HSCs). It is a determining step in the pathophysiology of non‐alcoholic steatohepatitis (NASH), an advanced form of fatty liver disease. The Hippo pathway and its effectors Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), have been implicated in hepatic fibrogenesis. However, how YAP and TAZ are regulated in HSCs remains incompletely understood. Here in this work, we have identified Sirtuin 6 (SIRT6), a NAD+ dependent deacetylase, as a regulator of YAP and TAZ in HSCs. Using primary HSCs deficient in SIRT6 from mice fed a moderately‐high‐fat‐cholesterol‐cholate diet (HFCC) for one week as well as in vitro models using transforming growth factor‐β (TGF‐β) treated LX‐2 cells, we have revealed that YAP and TAZ were acetylated during the HSC activation. Moreover, expression of YAP and TAZ target genes including connective tissue growth factor (Ctgf), cysteine‐rich angiogenic inducer 61 (Cyr61) and ankyrin repeat domain 1 (Ankrd1) was increased in those HSCs. Immunofluorescence microscopy analysis of liver sections obtained from Sirt6 HSC‐specific knockout mice fed HFCC diet for six weeks showed elevated YAP nuclear localization in the HSCs, suggesting an activation of YAP during fibrosis. Further in vitroanalysis of YAP/TAZ activity upon gain‐ and loss‐of‐function of SIRT6 in TGF‐β treated LX‐2 cells showed a decrease or increase of Ctgf, Cyr61 and Col3a1gene expression,respectively. To understand how SIRT6 regulates YAP and TAZ, we performed immunoprecipitation and lysine acetylation analysis, and our data showed that SIRT6 indeed interacted with YAP and TAZ and deacetylated them. Taken together, our study has identified a key regulatory axis in which SIRT6 interacts with YAP/TAZ and deacetylates them at several lysine residues, thereby suppressing their transcriptional co‐activator activity during fibrosis.

Extracellular Matrix Stiffness and TGFβ2 Regulate YAP/TAZ Activity in Human Trabecular Meshwork Cells.

Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGFβ2) levels in the aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGFβ2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ nuclear localization potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGFβ2 increased nuclear YAP/TAZ in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGFβ2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGFβ2-induced hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

Deubiquitinase JOSD2 stabilizes YAP/TAZ to promote cholangiocarcinoma progression

Cholangiocarcinoma (CCA) has emerged as an intractable cancer with scanty therapeutic regimens. The aberrant activation of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are reported to be common in CCA patients. However, the underpinning mechanism remains poorly understood. Deubiquitinase (DUB) is regarded as a main orchestrator in maintaining protein homeostasis. Here, we identified Josephin domain-containing protein 2 (JOSD2) as an essential DUB of YAP/TAZ that sustained the protein level through cleavage of polyubiquitin chains in a deubiquitinase activity-dependent manner. The depletion of JOSD2 promoted YAP/TAZ proteasomal degradation and significantly impeded CCA proliferation in vitro and in vivo. Further analysis has highlighted the positive correlation between JOSD2 and YAP abundance in CCA patient samples. Collectively, this study uncovers the regulatory effects of JOSD2 on YAP/TAZ protein stabilities and profiles its contribution in CCA malignant progression, which may provide a potential intervention target for YAP/TAZ-related CCA patients.

COMPRENDERE LE BASI MOLECOLARI DELLA DISABILITÀ INTELLETTIVA ATTRAVERSO IL MODELLO DEI NEURONI DI CELLULE STAMINALI PLURIPOTENTI INDOTTE

In 2012 the Nobel Prize for Medicine was awarded to Sir John Gurdon and Shinya Yamanaka for their joined discovery that mature, specialized cells can be reprogrammed to become immature pluripotent cells capable of developing into all different tissues of the body. This seminal breakthrough changed our vision of developmental biology and made possible to generate in vitro from somatic cells of any healthy or affected individual Induced Pluripotent Stem Cells (iPSCs) and differentiate them into virtually all types of cells. Scientists around the world were provided with the tool of modelling human diseases, even those involving inaccessible tissues such as the nervous system, to study the pathological process. iPSCs also revealed an enormous potential for pharmaceutical and clinical applications as on these cells a myriad of small molecules or candidate drugs can be tested some of which hopefully will become new effective medicines for intractable diseases. We have generated the first iPSC-derived neuronal model for the Rubinstein-Taybi syndrome (RSTS), a mendelian neurodevelopmental disorder, characterized by facial dysmorphisms, growth and speech delay, skeletal dysplasia and intellectual disability often associated to behavior disorder. RSTS is caused by mutation in the homologous CREBBP and EP300 genes encoding proteins acting as chromatin regulators through their intertwined activity of transcriptional co-activators and acetyltransferases (KATs) on histone and non histone proteins. I summarize herein the workflow used to generate iPSC-derived neurons (iNeurons) from six RSTS patients with a variable cognitive impairment in parallel to iNeurons from four healthy controls. Immunohistochemical characterization of samples at the stages of iPSCs, neural rosettes, neural progenitors and post-mitotic cortical neurons did not reveal gross alterations in the expression of stage-specific differentiation markers across patients’ or between patients’ and controls’ neuronal cultures. Conversely, altered morphology of patients’ differentiating neurons, showing reduced branch length and increased branch number and hypoexcitability of differentiated neurons emerged as relevant “disease” biomarkers. Both the anomalous neuronal morphology and the impaired electrophysiological performance varied across iNeurons from different RSTS patients. possibly reflecting cognitive and behavioural impairment of the donor patients. To validate the identified morpho-functional markers we performed further studies on iNeurons from the RSTS patient with the most severe intellectual disability by using trichostatin A (TSA), an epidrug that by inhibiting histonedeacetylases (HDAcs), the KATs opposing enzymes, has been successfully applied in different cbp+/cbp- mouse models with improvement of some neurological abnormalities. Short acute and chronic TSA treatment of two independent neural progenitor lines from the selected RSTS patient determined in both replicas a consistent reversal of a few morphological abnormalities and a significant rescue of the defective electrophysiological performance, highlighting the potential postnatal intervention of epidrugs to ameliorate the cognitive impairment of RSTS patients.

Современный этап нейропротекции - развитие митохондриально-направленных подходов

Обзор посвящен рассмотрению современных подходов к фармакологической модуляции структурно-функционального состояния митохондриального аппарата нейронов как перспективной стратегии терапии ишемического инсульта головного мозга, составляющего по современным оценкам до 85% всех случаев острого нарушения мозгового кровообращения. В обзоре проведен анализ классических представлений о нейропротекции как терапии, направленной на блокирование ключевых патогенетических звеньев «ишемического каскада», в контексте современного понимания приоритетной роли коррекции митохондриальной дисфункции в ограничении механизмов повреждения при церебральной ишемии. Обсуждаются возможности применения препаратов плейотропного нейропротекторного действия, реализующих митохондриально-направленные защитные эффекты через модуляцию активности транскрипционного коактиватора PGC-1α (peroxisome proliferator-activated receptor-1γ coactivator-1α), контролирующего процессы биогенеза митохондрий, ангиогенеза, ферментативное звено антиоксидантной системы. The review addresses modern approaches to pharmacological modulation of structure and function of the neuronal mitochondrial apparatus. This is a promising strategy for therapy of ischemic stroke, which accounts for up to 85% of all cases of acute cerebrovascular disease. The review analyzes classical concepts of neuroprotection as a therapy aimed at blocking key pathogenetic components of the “ischemic cascade”. These concepts are based on current ideas about the importance of correcting mitochondrial dysfunction for alleviation of damage in cerebral ischemia. The authors discussed possibilities of using pleiotropic neuroprotectors that implement mitochondrial-targeted protective effects by modulating the activity of transcriptional coactivator, peroxisome proliferator-activated receptor-1γ coactivator-1α (PGC-1α), which controls mitochondrial biogenesis, angiogenesis, and the enzymatic antioxidant system.

Lactate-fueled oxidative metabolism drives DNA methyltransferase 1-mediated transcriptional co-activator with PDZ binding domain protein activation.

Activity of transcriptional co‐activator with PDZ binding domain (TAZ) protein is strongly implicated in the pathogenesis of human cancer and is influenced by tumor metabolism. High levels of lactate concentration in the tumor microenvironment as a result of metabolic reprogramming are inversely correlated with patient overall survival. Herein, we investigated the role of lactate in the regulation of the activity of TAZ and showed that glycolysis‐derived lactate efficiently increased TAZ expression and activity in lung cancer cells. We showed that the reactive oxygen species (ROS) generated by lactate‐fueled oxidative phosphorylation (OXPHOS) in mitochondria activated AKT and thereby inhibited glycogen synthase kinase 3 beta/beta‐transducin repeat‐containing proteins (GSK‐3β/β‐TrCP)‐mediated ubiquitination and degradation of DNA methyltransferase 1 (DNMT1). Upregulation of DNMT1 by lactate caused hypermethylation of TAZ negative regulator of the LATS2 gene promoter, leading to TAZ activation. Moreover, TAZ binds to the promoter of DNMT1 and is necessary for DNMT1 transcription. Our study showed a molecular mechanism of DNMT1 in linking tumor metabolic reprogramming to the Hippo‐TAZ pathway and functional significance of the DNMT1‐TAZ feedback loop in the migratory and invasive potential of lung cancer cells.

Alternative isoforms of BmYki have different transcriptional co-activator activity in the silkworm, Bombyx mori.

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.

Cell type-dependent function of LATS1/2 in cancer cell growth.

The Hippo pathway controls organ size and tissue homeostasis, and its dysregulation often contributes to tumorigenesis. Extensive studies have shown that the Hippo pathway inhibits cell proliferation, and survival in a cell-autonomous manner. We examined the function of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in cancer cells. As expected, loss of LATS1/2 promotes cancer cell growth in most cell lines. Surprisingly, however, LATS1/2 deletion inhibits the growth of murine MC38 colon cancer cells, especially under detachment conditions. This growth inhibitory effect caused by LATS1/2 deletion is due to uncontrolled activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the key downstream transcriptional coactivators inhibited by LATS1/2. We identified Wnt inducible signaling pathway protein 2 (Wisp2) and coiled-coil domain containing 80 (Ccdc80) as direct targets of YAP/TAZ. Their expression is selectively induced by LATS1/2 deletion in MC38 cells. Furthermore, deletion of WISP2 and CCDC80 prevents the growth inhibitory effect of LATS1/2 loss in MC38 cells. Our study demonstrates that the function of LATS1/2 in cell growth is cell context dependent, suggesting that LATS1/2 inhibition can be a therapeutic approach for some cancer types.

Hippo pathway inhibition by blocking the YAP/TAZ–TEAD interface: a patent review

ABSTRACTIntroduction: The Hippo pathway represents a new and intriguing opportunity for the treatment of cancer. Activation or overexpression of Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ) has been shown to lead to cell transformation and tumor development. To date, no small molecule compounds targeting this pathway have progressed to the clinic, illustrating both its potential and its infancy.Areas covered: The present review seeks to summarize published patent applications from assignee companies that have disclosed direct small molecule inhibitors of the YAP/TAZ–transcriptional enhanced associate domain (TEAD) interaction.Expert opinion: The Hippo pathway, and specifically the YAP/TAZ–TEAD transcriptional complex, has been shown to be a promising target for the treatment of cancer. However, reports in the area of small molecules targeting the YAP/TAZ–TEAD transcriptional activation complex are few and far between, with only two published patent applications that disclose compounds with moderate levels of pathway inhibition. Interestingly, the YAP/TAZ–TEAD complex can be disrupted through two very different mechanisms, one of which is direct inhibition at either the Ω-loop or the α-helix of the YAP–TEAD binding interface. Both YAP protein segments have been shown to be important to TEAD binding. Alternatively, it has been reported that allosteric inhibition might be accomplished by binding the TEAD palmitoylation pocket, thus disrupting YAP binding and also native protein stabilization. The advantages and liabilities of disrupting the YAP/TAZ–TEAD complex through these two distinct mechanisms have yet to be fully elucidated, and it remains unclear which approach, if any, will generate the first clinical stage inhibitor of the Hippo pathway.

Abstract A084: Reprogramming of androgen receptor gene expression by ACK1/TNK2 mediated histone H4 Tyr88-phosphorylation promotes castration resistance

Abstract Aberrant androgen receptor (AR) transcriptional co-activator activity underlies the onset and progression of prostate cancer (PC) to castration-resistant (CRPC) state. While anti-androgens provide immediate palliative benefits by negating AR transcriptional activity, resistance inevitably develops—either by AR gene amplification, mutation, or expression of splice variants, such as AR-V7. To achieve complete remission, ablation of AR expression appears to be the key. However, targeted inhibition of AR and AR-V7 gene expression with small-molecule inhibitors has not yet been accomplished. Recently, we uncovered a novel chromatin-regulated mechanism of AR transcriptional regulation mediated by the tyrosine kinase ACK1 (also known as TNK2). We observed that ACK1 phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex is read by H4-Y88-phosphorylation and in turn deposits the transcriptionally activating H3K4-trimethyl marks, promoting AR transcription in androgen-deficient environment. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Interestingly, we observed that AR itself was modified by acetylation at Lys609 residue in enzalutamide-resistant PC cells, revealing an additional mechanisms underlying castration resistance. Overall, these data reveal previously undocumented epigenetic circuitry that not only sustains AR and AR-V7 expression, but also provides mechanistic insight into rapid acquisition of resistance to antiandrogens. Citation Format: Nupam Mahajan, Kiran Mahajan, Harshani Lawrence, Nicholas Lawrence. Reprogramming of androgen receptor gene expression by ACK1/TNK2 mediated histone H4 Tyr88-phosphorylation promotes castration resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A084.

EGF Receptor-Dependent YAP Activation Is Important for Renal Recovery from AKI.

Increasing evidence indicates that renal recovery from AKI stems from dedifferentiation and proliferation of surviving tubule epithelial cells. Both EGF receptor (EGFR) and the Hippo signaling pathway are implicated in cell proliferation and differentiation, and previous studies showed that activation of EGFR in renal proximal tubule epithelial cells (RPTCs) plays a critical role in recovery from ischemia-reperfusion injury (IRI). In this study, we explored RPTC activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ), two key downstream effectors of the Hippo pathway, and their potential involvement in recovery from AKI. We used immunofluorescence to examine YAP expression in kidney biopsy samples from patients with clinical AKI and controls (patients with minimal change disease). Studies of RPTC activation of YAP and TAZ used cultured human RPTCs that were exposed to hypoxia-reoxygenation as well as knockout mice (with inducible deletions of Yap, Taz, or both occurring specifically in RPTCs) that were subjected to bilateral IRI. YAP was activated in RPTCs in kidneys from post-AKI patients and post-IRI mouse kidneys. Inhibition of the interaction of YAP and the TEA domain (TEAD) transcription factor complex by verteporfin or conditional deletion of YAP in RPTCs delayed renal functional and structural recovery from IRI, whereas TAZ deletion had no effect. Activation of the EGFR-PI3K-Akt pathway in response to IRI signaled YAP activation, which promoted cell cycle progression. This study shows that EGFR-PI3K-Akt-dependent YAP activation plays an essential role in mediating epithelial cell regeneration during kidney recovery from AKI.

PI3K Positively Regulates YAP and TAZ in Mammary Tumorigenesis Through Multiple Signaling Pathways.

Breast cancer is a leading cause of death in women worldwide. Active mutations of PI3K catalytic subunit PIK3CA (e.g., H1047R) and amplification of its homolog PIK3CB are observed in a large number of breast cancers. In recent years, aberrant activation of Transcriptional coactivator with PDZ binding motif (TAZ) and its paralog Yes-associated protein (YAP) have also been found to be important for breast cancer development and progression. However, whether PI3K interacts with YAP/TAZ during mammary tumorigenesis is unknown. Through a systematic gain-of-function screen for kinases involved in mammary tumorigenesis, we identified PIK3CB as a transformation-inducing kinase in breast cells. We further determined that PIK3CB positively regulates YAP and TAZ to promote transformation and inhibit mammary cell death in vitro PIK3CB coexpression with TAZ, rather than PIK3CB or TAZ alone, in human MCF10A nontumorigenic mammary cells is sufficient for tumor formation in mice in vivo Interestingly, we also determined that PIK3CA-H1047R enhances YAP and TAZ activity in mammary tumorigenesis in vitro Mechanistically, the regulation of YAP/TAZ by both PIK3CA and PIK3CB occurs through multiple signaling pathways including LATS-dependent and LATS-independent pathways. Therefore, in this study, we determine that PI3K and YAP/TAZ interact to promote breast cancer cell transformation.Implications: This study provides the first evidence that the Hippo pathway effectors TAZ and YAP are critical mediators of PI3K-induced mammary tumorigenesis and synergistically function together with PI3K in transformation of mammary cells. These findings may provide a novel rationale for targeting YAP/TAZ alone or in combination with PI3K inhibitors for breast cancer therapy in the future. Mol Cancer Res; 16(6); 1046-58. ©2018 AACR.

The Wnt-β-catenin signaling regulated MRTF-A transcription to activate migration-related genes in human breast cancer cells.

MRTF-A is a transcriptional co-activator being critical for multiple processes including tissue fibrosis and cancer metastasis. The Rho-actin signaling stimulates the nuclear translocation and transcriptional activity of MRTF-A with little effect on the expression of MRTF-A gene. High expression of MRTF-A was observed in pancreatic cancer tissues and in TGF-β treated breast cancer cells. However, the mechanism for the upregulation of MRTF-A gene remains unclear. In this study, we showed that the transcription of MRTF-A was regulated by the Wnt-β-catenin signaling in breast cancer cells. LiCl treatment, Wnt3a treatment or β-catenin overexpression enhanced the transcription of MRTF-A gene. In agreement, depletion of β-catenin with siRNA diminished MRTF-A transcription. With ChIP assays, β-catenin was identified to interact with the MRTF-A promoter whereby it increased histone H4 acetylation and RNA polymerase II association. Further, results of RT-qPCR and Western-blotting supported that the transcriptional co-activator activity of MRTF-A was controlled by both the Rho-actin and the Wnt-β-catenin signaling pathways. MRTF-A was required for cell migration stimulated by the Wnt-β-catenin signaling. Taken together, our results suggest that MRTF-A integrates the Rho-actin and the Wnt-β-catenin signaling to regulate migration-related genes and consequently increases the mobility of breast cancer cells.

Abstract 5895: Targeting RhoA-regulated gene transcription in drug-resistant melanoma

Abstract Much of the recent focus of melanoma targeted therapy has been on the ERK pathway, which is aberrantly activated in approximately 90% of melanoma tumors. Over half of these express BRAFV600E. Current targeted therapies such as Vemurafenib (BRAFV600E inhibitor), or a combination therapy of BRAF + MEK inhibitors show profound initial effects in a majority of BRAFV600E expressing tumors. However, these responses are often short-lived and resistance typically develops within months. The goal of this work is to identify pharmacologically targetable resistance mechanisms so that effective combination therapies can be developed. Preliminary bioinformatics analysis suggests that the RhoA subfamily of Rho GTPases is activated in BRAFi-resistant cancer cell lines and BRAFi/MEKi-resistant human melanoma tumors. Thus, we hypothesize that RhoA promotes BRAFi resistance, so simultaneously targeting RhoA and BRAF may reverse drug resistance. Using cell line models of acquired BRAFi-resistance we demonstrated that the RhoA pathway is activated in a subset of resistant cell lines. The cell lines with increased RhoA activation have increased sensitivity to multiple ROCK inhibitors. In addition to regulating the cytoskeleton, RhoA can also regulate gene transcription through activation of multiple transcriptional co-activators, including MRTF and YAP. MRTF and YAP regulated gene transcription through their role as transcriptional co-activators and their interaction with chromatin remodeling complexes. MRTF and YAP are both activated in BRAFi-resistant cell lines. Indirectly targeting MRTF with CCG-222740 or YAP with the YES1 inhibitor Dasatinib effectively kills BRAFi-resistant melanoma cells. Sox10 was previously identified as a gene which promotes BRAFi resistance. Expression of Sox10 is downregulated, and Sox9 is upregulated, 100-1000-fold exclusively in the resistant cell lines which have RhoA activation. Further, a Sox10 gene signature is inversely correlated with a RhoA/C signature in the TCGA dataset and melanoma scRNA-seq data. Sox10 loss is a major drug resistance melanoma in melanoma. These data suggest that targeting RhoA-regulated gene transcription may be an effective mechanism to target Sox9High/Sox10Low cells, and ultimately prevent or reverse drug resistance. Taken together these data suggest that MRTF and YAP promote Vemurafenib resistance in Sox9High/Sox10Low melanoma cells. Citation Format: Sean A. Misek, Kathleen A. Gallo, Richard R. Neubig. Targeting RhoA-regulated gene transcription in drug-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5895.

Developing Spindlin1 small-molecule inhibitors by using protein microarrays.

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.