Regulation Of TAZ Research Articles

Tolfenamic acid negatively regulates YAP and TAZ expression in human cancer cells.

Several diseases are associated with improper regulation of the Hippo pathway, which plays an important role in cell proliferation and cancer metastasis. Overactivation of the YAP and TAZ proteins accelerates cell proliferation, invasion, and migration during tumorigenesis. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) that exhibits activity against various types of cancer. In this study, we observed that TA decreased YAP and TAZ protein levels in cancer cells. TA increased the phosphorylation of YAP and TAZ, leading to the degradation of YAP and TAZ in the cytoplasm and nucleus. TA predominantly affected multiple phosphodegron sites in the YAP and TAZ and lowered 14-3-3β protein expression, causing YAP and TAZ to enter the ubiquitination pathway. Proteins that affect YAP and TAZ regulation, such as NAG-1 and several YAP/TAZ E3 ligases, were not involved in TA-mediated YAP/TAZ degradation. In summary, our results indicate that TA affects phosphodegron sites on YAP/TAZ, demonstrating a novel effect of TA in tumorigenesis.

Loss of TAZ after YAP deletion severely impairs foregut development and worsens cholestatic hepatocellular injury.

We previously showed that loss of yes-associated protein 1 (YAP) in early liver development (YAPKO) leads to an Alagille syndrome-like phenotype, with failure of intrahepatic bile duct development, severe cholestasis, and chronic hepatocyte adaptations to reduce liver injury. TAZ, a paralog of YAP, was significantly upregulated in YAPKO hepatocytes and interacted with TEA domain family member (TEAD) transcription factors, suggesting possible compensatory activity. We deleted both Yap1 and Wwtr1 (which encodes TAZ) during early liver development using the Foxa3 promoter to drive Cre expression, similar to YAPKO mice, resulting in YAP/TAZ double knockout (DKO) and YAPKO with TAZ heterozygosity (YAPKO TAZHET). We evaluated these mice using immunohistochemistry, serum biochemistry, bile acid profiling, and RNA sequencing. DKO mice were embryonic lethal, but their livers were similar to YAPKO, suggesting an extrahepatic cause of death. Male YAPKO TAZHET mice were also embryonic lethal, with insufficient samples to determine the cause. However, YAPKO TAZHET females survived and were phenotypically similar to YAPKO mice, with increased bile acid hydrophilicity and similar global gene expression adaptations but worsened the hepatocellular injury. TAZ heterozygosity in YAPKO impacted the expression of canonical YAP targets Ctgf and Cyr61, and we found changes in pathways regulating cell division and inflammatory signaling correlating with an increase in hepatocyte cell death, cell cycling, and macrophage recruitment. YAP loss (with or without TAZ loss) aborts biliary development. YAP and TAZ play a codependent critical role in foregut endoderm development outside the liver, but they are not essential for hepatocyte development. TAZ heterozygosity in YAPKO livers increased cell cycling and inflammatory signaling in the setting of chronic injury, highlighting genes that are especially sensitive to TAZ regulation.

A positive feedback loop of the TAZ/β-catenin axis promotes Helicobacter pylori-associated gastric carcinogenesis.

Helicobacter pylori infection is the strongest known risk factor for gastric cancer. The Hippo signaling pathway controls organ size and maintains tissue homeostasis by coordinately regulating cell growth and proliferation. Here, we demonstrate the interactive role of TAZ, the transcriptional coactivator of the Hippo pathway, and beta-catenin in promoting the pathogenesis of H. pylori infection. TAZ expression was evaluated in human gastric tissues and H. pylori-infected insulin-gastrin (INS-GAS) mice. Western blot, immunofluorescence, immunohistochemistry, and RT-PCR assays were performed. Coimmunoprecipitation was performed to examine the interaction between TAZ and β-catenin. TAZ and β-catenin were silenced using small interfering RNAs. HA-β-catenin and Flag-TAZ were constructed. Increased TAZ was noted in human gastric cancer tissues compared to chronic gastritis tissues and in H. pylori-positive gastritis tissues compared to H. pylori-negative gastritis tissues. In addition, H. pylori infection induced TAZ expression and nuclear accumulation in the gastric tissue of INS-GAS mice and cultured gastric epithelial cells, which was dependent on the virulence factor CagA. Moreover, TAZ or β-catenin knockdown significantly suppressed H. pylori infection-induced cell growth, survival, and invasion. Furthermore, the interactive regulation of TAZ and β-catenin activation was revealed. Finally, β-catenin was required for H. pylori-induced TAZ activation. These findings suggest the existence of a positive feedback loop of activation between TAZ and β-catenin that could play an important role in CagA+ H. pylori infection-induced gastric carcinogenesis. TAZ inhibition represents a potential target for the prevention of H. pylori infection-associated gastric cancer.

Regulation of TAZ by DEPTOR controls mesenchymal progenitors lineage commitment in response to PTH1R signaling

Regulation of TAZ by DEPTOR controls mesenchymal progenitors lineage commitment in response to PTH1R signaling

Abstract A16: The α-arrestin ARRDC3 functions as a metastasis suppressor by regulating GPCR activation of the Hippo pathway

Abstract G protein-coupled receptors (GPCRs) are key regulators of the Hippo pathway, a well-established mediator of tumorigenesis and cancer progression. The Hippo pathway is a promising therapeutic target, yet developing inhibitors for the core pathway components has proven difficult. As critical regulators of the Hippo pathway, GPCRs represent a potential upstream therapeutic target for cancer. However, despite the success and promise of GPCRs as therapeutic targets, there are currently no FDA-approved drugs targeting GPCRs for cancer. Protease-activated receptor-1 (PAR1) is a GPCR that has been shown to activate the Hippo pathway and also promote breast cancer progression. PAR1 is overexpressed in breast cancer patient tissue biopsies and in breast carcinoma cell lines and correlates with increased rates of metastasis and poor prognosis and increased invasion and metastatic potential, respectively. One mechanism that leads to PAR1 aberrant overexpression is defective lysosomal trafficking and degradation of the receptor, leading to persistent signaling. We recently showed that arrestin domain containing protein-3 (ARRDC3), an adaptor protein for E3 ubiquitin ligases, functions as a tumor suppressor by controlling proper PAR1 trafficking and degradation in highly aggressive, triple-negative breast carcinoma cells. The dysregulation of PAR1 trafficking due to loss of ARRDC3 expression leads to persistent signaling and promotes breast cancer invasion. However, the mechanism by which ARRDC3 specifically regulates PAR1-stimulated Hippo signaling to promote breast cancer metastasis remains unknown. Here, we show that ARRDC3 regulates GPCR activation of the Hippo pathway in basal-like breast cancer, where the transcriptional coactivators YAP and TAZ play differential roles in GPCR-mediated signaling and invasive capacity. ARRDC3 re-expression in invasive breast carcinoma cells attenuates PAR1-mediated activation of the Hippo pathway through regulation of TAZ but not YAP activation. Furthermore, siRNA knockdown of TAZ inhibits PAR1-stimulated Hippo signaling and invasion, whereas YAP siRNA knockdown has no effect. Our studies suggest a crucial role for ARRDC3 and TAZ in PAR1-Hippo pathway signaling in breast carcinoma invasion and metastasis. A better understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer. Citation Format: Aleena K.S. Arakaki, Wen-An Pan, Helen Wedegaertner, JoAnn Trejo. The α-arrestin ARRDC3 functions as a metastasis suppressor by regulating GPCR activation of the Hippo pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A16.

Glucose promotes epithelial‐mesenchymal transitions in bladder cancer by regulating the functions of YAP1 and TAZ

Glucose levels and type 2 diabetes (T2D) are both associated with tumorigenesis and epithelial‐mesenchymal transitions (EMTs). EMTs facilitate bladder cancer (BC) metastasis development, but the mechanism by which high‐glucose levels promote these EMTs in BC remains unclear. Therefore, we sought to elucidate the mechanism underlying EMT promotion due to increased glucose levels. T24 and UMUC‐3 cells were cultured in media containing different glucose concentrations. YAP1, TAZ, GLUT1 and EMT‐associated marker expression was analysed via Western blotting and qPCR. BC cell proliferation and invasion were assessed using MTT and Transwell assays, respectively. A xenograft nude mouse model of diabetes was used to evaluate tumour growth and metastasis in vivo. T2D was positively associated with pathologic grade (P = .016) and TNM stage (P < .001) in BC. High glucose triggered BC cell proliferation and invasion in both in vitro and in vivo conditions. High‐glucose levels also promoted EMTs in BC cells and increased YAP1 and TAZ expression. YAP1 or TAZ knockdown altered EMT marker expression and decreased GLUT1 expression. Overall, our results suggest that high‐glucose levels promote EMTs in BC cells via YAP1 and TAZ regulation. These effector molecules may be promising therapeutic targets for BC cases comorbid with T2D.

Isoprenylcysteine carboxylmethyltransferase is required for the impact of mutant KRAS on TAZ protein level and cancer cell self-renewal

Cancer stem cells possess the capacity for self-renewal and resistance to chemotherapy. It is therefore crucial to understand the molecular regulators of stemness in the quest to develop effective cancer therapies. TAZ is a transcription activator that promotes stem cell functions in post-development mammalian cells; suppression of TAZ activity reduces or eliminates cancer stemness in select cancers. Isoprenylcysteine carboxylmethyltransferase (ICMT) is the unique enzyme of the last step of posttranslational prenylation processing pathway that modifies several oncogenic proteins, including RAS. We found that suppression of ICMT results in reduced self-renewal/stemness in KRAS-driven pancreatic and breast cancer cells. Silencing of ICMT led to significant reduction of TAZ protein levels and loss of self-renewal ability, which could be reversed by overexpressing mutant KRAS, demonstrating the functional impact of ICMT modification on the ability of KRAS to control TAZ stability and function. Contrary to expectation, YAP protein levels appear to be much less susceptible than TAZ to the regulation by ICMT and KRAS, and YAP is less consequential in regulating stemness characteristics in these cells. Further, we found that the ICMT-dependent KRAS regulation of TAZ was mediated through RAF, but not PI3K, signaling. Functionally, we demonstrate that a signaling cascade from ICMT modification of KRAS to TAZ protein stability supports cancer cell self-renewal abilities in both in vitro and in vivo settings. In addition, studies using the proof-of-concept small molecule inhibitors of ICMT confirmed its role in regulating TAZ and self-renewal, demonstrating the potential utility of targeting ICMT to control aggressive KRAS-driven cancers.

The regulation of Yorkie, YAP and TAZ: new insights into the Hippo pathway.

The Hippo pathway is a highly conserved signalling pathway that regulates multiple biological processes, including organ size control and cell fate. Since its discovery, genetic and biochemical studies have elucidated several key signalling steps important for pathway activation and deactivation. In recent years, technical advances in microscopy and genome modification have allowed new insights into Hippo signalling to be revealed. These studies have highlighted that the nuclear-cytoplasmic shuttling behaviour of the Hippo pathway transcriptional co-activators Yorkie, YAP and TAZ is far more dynamic than previously appreciated, and YAP and TAZ are also regulated by liquid-liquid phase separation. Here, we review our current understanding of Yorkie, YAP and TAZ regulation, with a focus on recent microscopy-based studies.

Polycystin 1 is an atypical adhesion GPCR that responds to non‐canonical WNT signals and inhibits GSK3β

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, which encode PC1 and PC2, respectively. PC1 is a 460kD multi‐spanning membrane protein. PC1 undergoes multiple proteolytic cleavages, one of which is an autocatalytic cleavage that produces a large N terminal fragment (NTF) that remains non‐covalently attached to the membrane‐embedded C terminal fragment (CTF). A recent study demonstrates that PC1 serves as a receptor for Wnt ligands through the direct binding of these ligands to the PC1 NTF. We find that PC1 responds to Wnt9b by shedding its 350kD NTF, which bears a striking resemblance to the activation mechanism of an adhesion GPCR. For aGPCRs, NTF removal liberates a short peptide sequence near the GPS cleavage site called stachel, which then interacts with an internal binding site and engages a G protein signalling cascade. We find that Wnt9b induces a PC1‐dependent phosphorylation and inhibition of the GSK3β kinase through the RhoA GTPase‐ROCK kinase pathway. Expression of a “constitutively active” aGPCR form of PC1 that lacks its NTF leads to profound suppression of GSK3β activity that is dependent upon RhoA and ROCK. Interestingly, GSK3β is an important negative regulator of the HIPPO/non‐canonical Wnt signaling target TAZ(WWTR1), lack of which leads to the development severe renal cysts. Furthermore, recent data show that pharmacological inhibition of GSK3β is beneficial in mouse models of ADPKD. We find that exogenous expression of an active form of TAZ in PC1 null cells or in PKD1a/b morphant zebrafish suppresses the development of relevant phenotypes. We also demonstrate that pharmacological inhibition of GSK3β leads to accumulation of the TAZ protein and to reduced cystogenesis in a 3D matrigel assay employing PC1 null cells. TAZ abundance and activity have been shown to be upregulated through the non‐canonical Wnt signalling pathway. We find that HEK293 cells that express PC1 and PC2 respond to Wnt9b treatment by significantly increasing TAZ abundance as compared to the wild type HEK293 cells treated in the same manner. Taken together, our data suggest that PC1 is an aGPCR‐like receptor for non‐canonical Wnt ligands that participates in a novel signalling pathway by linking non‐canonical Wnt ligands to the GSK3β‐dependent regulation of TAZ, a multifaceted signalling molecule whose absence is sufficient to induce renal cystic disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunofluorescence Microscopy to Study Endogenous TAZ in Mammalian Cells.

The transcriptional coactivator with PDZ-binding motif (TAZ), which is encoded by the WWTR1 gene, is a key transcriptional effector of the Hippo signaling pathway. TAZ function has been implicated in a variety of developmental processesand diseases, most notably in drivingoncogenesis. Given that nuclear-cytoplasmic localization dynamics dictate TAZ activity, techniques for visualizing TAZ localization are critical for its study. Here we describe an immunofluorescence microscopy protocol that allows for the visualization of TAZ subcellular localization in mammalian cells, offering an approach that can aid in the analysis of TAZ regulation and function.

STAT1-mediated upregulation of lncRNA LINC00174 functions a ceRNA for miR-1910-3p to facilitate colorectal carcinoma progression through regulation of TAZ

Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in colorectal carcinoma (CRC) remains poorly understood. In present study, we aim to investigate the role of lncRNA LINC00174 in CRC carcinogenesis. We observed that increased expression of LINC00174 in CRC tissues and cells in comparison to their corresponding controls. Moreover, the aberrant overexpression of LINC00174 indicated the poor prognosis of CRC patients. Silence of LINC00174 was able to repress CRC cell growth in vitro and in vivo. We first reported that transcription factor STAT1 mediated LINC00174 expression in CRC. In addition, rescue assay was performed to further confirm that LINC00174 contributed to CRC progression by regulating miR-1910-3p/TAZ signal pathway. Taken together, our study discovered the oncogenic role of LINC00174 in clinical specimens and cellular experiments, showing the potential LINC00174/miR-1910-3p/TAZ pathway. This results and findings provide a novel insight for CRC tumorigenesis.

TGF-β1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism

Hippo pathway transcriptional coactivators TAZ and YAP and the TGF-β1 (TGFβ) effector Smad3 regulate a common set of genes, can physically interact, and exhibit multilevel cross-talk regulating cell fate-determining and fibrogenic pathways. However, a key aspect of this cross-talk, TGFβ-mediated regulation of TAZ or YAP expression, remains uncharacterized. Here, we show that TGFβ induces robust TAZ but not YAP protein expression in both mesenchymal and epithelial cells. TAZ levels, and to a lesser extent YAP levels, also increased during experimental kidney fibrosis. Pharmacological or genetic inhibition of Smad3 did not prevent the TGFβ-induced TAZ up-regulation, indicating that this canonical pathway is dispensable. In contrast, inhibition of p38 MAPK, its downstream effector MK2 (e.g. by the clinically approved antifibrotic pirferidone), or Akt suppressed the TGFβ-induced TAZ expression. Moreover, TGFβ elevated TAZ mRNA in a p38-dependent manner. Myocardin-related transcription factor (MRTF) was a central mediator of this effect, as MRTF silencing/inhibition abolished the TGFβ-induced TAZ expression. MRTF overexpression drove the TAZ promoter in a CC(A/T-rich)6GG (CArG) box-dependent manner and induced TAZ protein expression. TGFβ did not act by promoting nuclear MRTF translocation; instead, it triggered p38- and MK2-mediated, Nox4-promoted MRTF phosphorylation and activation. Functionally, higher TAZ levels increased TAZ/TEAD-dependent transcription and primed cells for enhanced TAZ activity upon a second stimulus (i.e. sphingosine 1-phosphate) that induced nuclear TAZ translocation. In conclusion, our results uncover an important aspect of the cross-talk between TGFβ and Hippo signaling, showing that TGFβ induces TAZ via a Smad3-independent, p38- and MRTF-mediated and yet MRTF translocation-independent mechanism.

TGFβ-TAZ/SRF signalling regulates vascular smooth muscle cell differentiation.

Vascular smooth muscle cells (VSMCs) do not terminally differentiate; they modulate their phenotype between proliferative and differentiated states, which is a major factor contributing to vascular diseases. TGFβ signalling has been implicated in inducing VSMC differentiation, although the exact mechanism remains largely unknown. Our goal was to assess the network of transcription factors involved in the induction of VSMC differentiation, and to determine the role of TAZ in promoting the quiescent VSMC phenotype. TGFβ robustly induces VSMC marker genes in 10T1/2 mouse embryonic fibroblast cells and the potent transcriptional regulator TAZ has been shown to retain Smad complexes on DNA. Thus, the role of TAZ in regulation of VSMC differentiation was studied. Using primary aortic VSMCs coupled with siRNA-mediated gene silencing, our studies reveal that TAZ is required for TGFβ induction of smooth muscle genes and is also required for the differentiated VSMC phenotype; synergy between TAZ and SRF, and TAZ and Myocardin (MyoC856), in regulating smooth muscle gene activation was observed. These data provide evidence of components of a novel signalling pathway that links TGFβ signalling to induction of smooth muscle genes through a mechanism involving regulation of TAZ and SRF proteins. In addition, we report a physical interaction of TAZ and MyoC856. These observations elucidate a novel level of control of VSMC induction which may have implications for vascular diseases and congenital vascular malformations.

Regulation of TAZ in cancer

TAZ, a transcriptional coactivator with PDZ-binding motif, is encoded by WWTR1 gene (WW domain containing transcription regulator 1). TAZ is tightly regulated in the hippo pathway-dependent and -independent manner in response to a wide range of extracellular and intrinsic signals, including cell density, cell polarity, F-actin related mechanical stress, ligands of G protein-coupled receptors (GPCRs), cellular energy status, hypoxia and osmotic stress. Besides its role in normal tissue development, TAZ plays critical roles in cell proliferation, differentiation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT), and stemness in multiple human cancers. We discuss here the regulators and regulation of TAZ. We also highlight the tumorigenic roles of TAZ and its potential therapeutic impact in human cancers.

Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ)

Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway.

MRTF/SRF dependent transcriptional regulation of TAZ in breast cancer cells.

Dysregulation of Hippo pathway results in activation of transcriptional co-activators YAP/TAZ in breast cancer. Previously, we showed that overexpression of TAZ in breast cancer promotes cell migration, invasion and tumorigenesis. Here, we show that upregulation of TAZ in breast cancers could also be due to dysregulation of TAZ transcription. Heregulin β1 (HRG1) increases TAZ mRNA level in breast cancer cells. TAZ is a direct target of MRTF/SRF transcriptional factors which are activated by HRG1. Both MRTF/SRF and TAZ are the important downstream effectors enhancing cell migration induced by HRG1. TAZ mRNA level is correlated with nuclear localization of MRTF in breast cancer cells and the mRNA level of MRTF/SRF direct target genes in breast cancers, indicating the correlation between MRTF/SRF activity and TAZ expression. Our results provide new insights into the transcriptional regulation of TAZ and dysregulation mechanism of TAZ in breast cancer, which could be a new therapeutic strategy for breast cancer.

TAZ Protein Accumulation Is Negatively Regulated by YAP Abundance in Mammalian Cells

The mammalian Hippo signaling pathway regulates cell growth and survival and is frequently dysregulated in cancer. YAP and TAZ are transcriptional coactivators that function as effectors of this signaling pathway. Aberrant YAP and TAZ activity is reported in several human cancers, and normally the expression and nuclear localization of these proteins is tightly regulated. We sought to establish whether a direct relationship exists between YAP and TAZ. Using knockdown and overexpression experiments we show YAP inversely regulates the abundance of TAZ protein by proteasomal degradation. Interestingly this phenomenon was uni-directional since TAZ expression did not affect YAP abundance. Structure/function analyses suggest that YAP-induced TAZ degradation is a consequence of YAP-targeted gene transcription involving TEAD factors. Subsequent investigation of known regulators of TAZ degradation using specific inhibitors revealed a role for heat shock protein 90 and glycogen synthase kinase 3 but not casein kinase 1 nor LATS in YAP-mediated TAZ loss. Importantly, this phenomenon is conserved from mouse to human; however, interestingly, different YAP isoforms varied in their ability to degrade TAZ. Since shRNA-mediated TAZ depletion in HeLa and D645 cells caused apoptotic cell death, we propose that isoform-specific YAP-mediated TAZ degradation may contribute to the contradicting roles reported for YAP overexpression. This study identifies a novel mechanism of TAZ regulation by YAP, which has significant implications for our understanding of Hippo pathway regulation, YAP-isoform specific signaling, and the role of these proteins in cell proliferation, apoptosis, and tumorigenesis.

PARD3 induces TAZ activation and cell growth by promoting LATS1 and PP1 interaction.

The Hippo pathway plays a major role in organ size control, and its dysregulation contributes to tumorigenesis. The major downstream effectors of the Hippo pathway are the YAP/TAZ transcription co-activators, which are phosphorylated and inhibited by the Hippo pathway kinase LATS1/2. Here, we report a novel mechanism of TAZ regulation by the tight junction protein PARD3. PARD3 promotes the interaction between PP1A and LATS1 to induce LATS1 dephosphorylation and inactivation, therefore leading to dephosphorylation and activation of TAZ. The cytoplasmic, but not the tight junction complex associated, PARD3 is responsible for TAZ regulation. Our study indicates a potential molecular basis for cell growth-promoting function of PARD3 by modulating the Hippo pathway signaling in response to cell contact and cell polarity signals.

Hypoxia inducible factor-1 is activated by transcriptional co-activator with PDZ-binding motif (TAZ) versus WWdomain-containing oxidoreductase (WWOX) in hypoxic microenvironment of bone metastasis from breast cancer

The hypoxic microenvironment of bone marrow favours the bone metastasis process. Hypoxia inducible factor (HIF)-1α is hallmark for hypoxia, correlating with poor prognosis and radio/chemotherapy resistance of primary-breast carcinoma. For bone metastasis, the molecular mechanisms involved in HIF-1α expression and HIF-1 (α/β heterodimer)-transcription factor activity are scarcely known. We studied the role played by HIF-1 in the cross-talk between neoplastic and supportive-microenvironmental cells. Also, WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ) were taken into consideration evaluating whether these Hippo-pathway effectors affect bone-metastatic phenotype through HIF-1 activity. Considering bone-metastasis specimens, nuclear HIF-1α–TAZ co-localisation occurred in neoplastic and supportive cells, such as fibroblasts and endotheliocytes. Based on these data, the functional importance was verified using 1833-bone metastatic clone under hypoxia: nuclear HIF-1α and TAZ expression increased and co-immunoprecipitated, activating HIF-1-DNA binding and transactivation. In contrast, Wwox localised at perinuclear level in neoplastic cells of bone metastasis, being almost absent in supportive cells, and Wwox-protein expression diminished in hypoxic-1833 cells. Thus, TAZ regulation of HIF-1 activity might be important for bone-secondary growth, participating in metastasis-stroma cross-talk. Further, TAZ and HIF-1α-protein levels seemed correlated. In fact, blocking cyclooxygenase-2 with NS398 in hypoxic-1833 cells, not only HIF-1α decreased but also molecular-mechanism(s) upstream of the Hippo pathway were triggered: LATS-dependent TAZ phosphorylation seemed responsible for TAZ nucleus/cytoplasm translocation and degradation. In the 1833-xenograft model, NS398 largely prevented the outgrowth of bone-metastatic cells, probably related to remarkable-extracellular matrix assembly. We gained clinical insight into HIF-1α and TAZ as candidate biomarkers for bone avidity, relevant for early-therapeutic intervention against bone metastasis.

The N-terminal Phosphodegron Targets TAZ/WWTR1 Protein for SCFβ-TrCP-dependent Degradation in Response to Phosphatidylinositol 3-Kinase Inhibition

The Hippo tumor suppressor pathway plays a major role in development and organ size control, and its dysregulation contributes to tumorigenesis. TAZ (transcriptional co-activator with PDZ-binding motif; also known as WWTR1) is a transcription co-activator acting downstream of the Hippo pathway, and increased TAZ protein levels have been associated with human cancers, such as breast cancer. Previous studies have shown that TAZ is inhibited by large tumor suppressor (LATS)-dependent phosphorylation, leading to cytoplasmic retention and ubiquitin-dependent degradation. The LATS kinase, a core component of the Hippo pathway, phosphorylates the C-terminal phosphodegron in TAZ to promote its degradation. In this study, we have found that the N-terminal phosphodegron of TAZ also plays a role in TAZ protein level regulation, particularly in response to different status of cellular PI3K signaling. GSK3, which can be inhibited by high PI3K via AKT-dependent inhibitory phosphorylation, phosphorylates the N-terminal phosphodegron in TAZ, and the phosphorylated TAZ binds to β-TrCP subunit of the SCF(β-TrCP) E3 ubiquitin ligase, thereby leading to TAZ ubiquitylation and degradation. We observed that the TAZ protein level is elevated in tumor cells with high PI3K signaling, such as in PTEN mutant cancer cells. This study provides a novel mechanism of TAZ regulation and suggests a role of TAZ in modulating tissue growth and tumor development in response to PI3K signaling.