Ezrin Activation Research Articles

Heat shock protein family B member 1 facilitates ezrin activation to control cell migration in esophageal squamous cell carcinoma.

Ezrin plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC), providing a link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and protrusions. However, the mechanism by which ezrin is activated still remains unknown in ESCC. Here, we identify a novel interaction between ezrin and heat shock protein family B (small) member 1 (HSPB1) in ESCC cells by mass spectroscopy and co-immunoprecipitation. HSPB1 only interacts with inactive ezrin and binds to the α-helical coiled coil region of ezrin. Knockdown of HSPB1 resulted to the decline of phosphorylation at ezrin Thr567, markedly suppressing the ability of ezrin to bind to the actin cytoskeleton and migration of ESCC cells. Furthermore, neither the constitutively active phosphomimetic ezrin T567D, nor inactivated ezrin T567A could restore cell migration following HSPB1 knockdown. Low HSPB1 expression was associated with favorable overall survival of ESCC patients. Taken together, HSPB1, as an important partner, participates in the activation of ezrin and merits further evaluation as a novel therapeutic target against human ESCC.

P2.03-19 RET-Mediated Activation of Ezrin is Associated with Cell Motility and Survival in a Subset of Lung Adenocarcinomas

P2.03-19 RET-Mediated Activation of Ezrin is Associated with Cell Motility and Survival in a Subset of Lung Adenocarcinomas

TGFβ and IGF1R signaling activates protein kinase A through differential regulation of ezrin phosphorylation in colon cancer cells

Aberrant cell survival plays a critical role in cancer progression and metastasis. We have previously shown that ezrin, a cAMP-dependent protein kinase A-anchoring protein (AKAP), is up-regulated in colorectal cancer (CRC) liver metastasis. Phosphorylation of ezrin at Thr-567 activates ezrin and plays an important role in CRC cell survival associated with XIAP and survivin up-regulation. In this study, we demonstrate that in FET and GEO colon cancer cells, knockdown of ezrin expression or inhibition of ezrin phosphorylation at Thr-567 increases apoptosis through protein kinase A (PKA) activation in a cAMP-independent manner. Transforming growth factor (TGF) β signaling inhibits ezrin phosphorylation in a Smad3-dependent and Smad2-independent manner and regulates pro-apoptotic function through ezrin-mediated PKA activation. On the other hand, ezrin phosphorylation at Thr-567 by insulin-like growth factor 1 receptor (IGF1R) signaling leads to cAMP-dependent PKA activation and enhances cell survival. Further studies indicate that phosphorylated ezrin forms a complex with PKA RII, and dephosphorylated ezrin dissociates from the complex and facilitates the association of PKA RII with AKAP149, both of which activate PKA yet lead to either cell survival or apoptosis. Thus, our studies reveal a novel mechanism of differential PKA activation mediated by TGFβ and IGF1R signaling through regulation of ezrin phosphorylation in CRC, resulting in different cell fates. This is of significance because TGFβ and IGF1R signaling pathways are well-characterized tumor suppressor and oncogenic pathways, respectively, with important roles in CRC tumorigenesis and metastasis. Our studies indicate that they cross-talk and antagonize each other's function through regulation of ezrin activation. Therefore, ezrin may be a potential therapeutic target in CRC.

Glypican 3 (GPC3)‐CD81 axis regulates Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop optimum targeted therapies because of multiple pathways involved in the solid tumor carcinogenesis. We have shown that activation of CD81 is associated with phosphorylation of Ezrin, which is overexpressed in many types of solid tumors and has been identified as metastasis related protein for many years. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and liver regeneration by inhibiting CD81‐mediated p‐Ezrin/Hippo/Yap pathway. It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by different kinases. Since we have demonstrated the predominant GPC+++/CD81− pattern in HCC, the purpose of this study is to explore the regulation of expression and phosphorylation of Ezrin in HCC. We have found that CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell lines have dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity. Mice overexpressing GPC3 had decreased p‐Ezrin but increased Hippo activity. In mice with post‐natal Met knock out there was less Yap expression at baseline level. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated p‐Ezrin. Treatment with 20 umol of Ezrin inhibitor led to significant cell death selectively in JM2 cells in vitro.ConclusionsBoth HGF/Met and CD81 mediate p‐Ezrin activity and enhance nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human hepatocytes, GPC3 inhibits the CD81‐mediated p‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies. And Ezrin is a promising target of anti‐HCC therapies including primary lesion and metastasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ezrin/NF-kB activation regulates epithelial- mesenchymal transition induced by EGF and promotes metastasis of colorectal cancer

There is growing evidence that epithelial mesenchymal-transition (EMT) plays significant roles in terms of tumor metastasis. There are a lot of cytokines inducing EMT of tumor cells, EGF is one of the important cytokines.Ezrin is a connexin between the cytoskeleton and the cell membrane, which is closely related to the morphological movement and metastasis of tumor cells.EGF can activate Ezrin and affects cell motility. In recent years, many studies have shown that NF-kB acts as an important transcription factor, involving in the process of EMT. However, does Ezrin participate in the regulation of EGF-induced EMT through the NF-kB pathway? This question needs us to discuss.In the present study, we found that EGF could induce colorectal cancer cells to develop EMT,enhance their ability to invade and migrate and promotes phosphorylation of Ezrin Tyr353.On the other hand, inhibition of Ezrin could reverse EGF-induced EMT and inhibit NF-kB P65 translocating into the nucleus. Finally, knockout of Ezrin inhibited EGF-induced lung metastasis of colorectal cancer xenografts and abnormal activation of Ezrin and NF-kB were related with colorectal cancer metastasis and poor prognosis. Our present results suggest that Ezrin/NF-kB pathway may provide experimental evidence for new targeted drugs for colorectal cancer metastasis.

Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism.

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.

Glypican 3 (GPC3)‐CD81 axis regulates spleen tyrosine kinase(Syk)‐Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop better targeted therapies because of multiple pathways involve in the solid tumor carcinogenesis. GPC3 is over‐expressed in HCC. Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and GPC3 transgenic mice with targeted expression in hepatocytes have decreased liver regeneration. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Studies elsewhere have shown that activation of CD81 is associated with phosphorylation of Ezrin. The latter has been shown to regulate the Hippo pathway by modulation activity of tumor suppressor gene NF2 (Merlin), responsible for regulating nuclear levels of the protein known as Yes‐associated protein (Yap). It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by unknown mechanisms. Expression of Yap in the nucleus enhances hepatocyte growth. Mice over‐expressing GPC3 have decreased levels of nuclear Yap. Since we have demonstrated that GPC3 binds to CD81, the purpose of this study is to explore the role of GPC3 in regulation of Yap via CD81 and Hippo pathway and to investigate the possible mechanisms. CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell line had dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity in a Syk dependent manner. While mice over‐expressing GPC3 decreased p‐Ezrin but increased Hippo activity. Transgenic Met knock out mice showed less Yap expression at baseline level, and abrogated up regulated Yap expression which was observed in wild type mice after 2d PHx and increased subsequent apoptosis. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated Ezrin activity in vitro. Force expression of Syk expression in JM2 rat hepatoma cells downregulated Hippo activity while upregulated Ezrin activity in vitro.ConclusionsBoth Met‐phosphoEzrin and CD81‐phosphoEzrin signaling enhances nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human HCC, loss of CD81 expression and highly activity of HGF/Met promote the CD81‐phosphoEzrin effect on Hippo pathway and cause a increase in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies.

Abstract 2446: Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis

Abstract Ezrin is a member of the ezrin, radixin, moesin (ERM) protein family of membrane-cytoskeleton linkers. Ezrin has been implicated in many essential cellular functions including cell adhesion, motility, maintenance and determination of cell shape, cell proliferation and apoptosis, regulation of ion channels, morphogenesis and signal transduction. Ezrin promotes invasive and migratory capabilities of cancer cells. A high level of ezrin expression is associated with poor clinical outcome and metastatic behavior of pediatric solid tumors including osteosarcoma and rhabdomyosarcoma as well as multiple other tumor types. Ezrin, therefore, could be a promising molecular target for the prevention and treatment of cancer metastasis. We previously discovered two small molecule inhibitors, NSC305787 and NSC668394, which bind directly to ezrin and inhibit its activity in mediating the invasive phenotype of osteosarcoma cells in multiple in vitro and in vivo assays. In this study, we expand on our previous findings by demonstrating that NSC305787-treatment but not NSC668394 significantly reduces pulmonary metastasis in a genetically engineered mouse model of osteosarcoma. We assessed the pharmacokinetics of compounds in mice and demonstrated that NSC305787 has a more favorable pharmacokinetic profile compared with NSC668394. In order to uncover ezrin-mediated biological pathways that can be used for a specific pharmacodynamic marker(s) of response to ezrin inhibition, we profiled global gene expression in osteosarcoma cells after treatment with inhibitors. We identified several commonly up-regulated genes with functional relevance to integrated stress response, implicating that a common underlying mechanism may be shared by these compounds. We further validated the microarray data through extensive testing using real-time qPCR and verified the specificity of the transcriptional response using another novel ezrin inhibitor MMV667492 that we have identified recently from the MMV400 “Malaria Box” library. The effect of ezrin inhibitors on the expression of stress genes was recapitulated by siRNA-mediated depletion of ezrin. The up-regulation of stress genes was much weaker in cells with reduced ezrin levels compared to wild-type cells, indicating the specificity of the compounds on ezrin-mediated cellular responses. Analysis of the expression of stress genes in white blood cells and skin of NSC305787-treated mice demonstrated up-regulation of the DDIT4/REDD1, suggesting that DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of response to ezrin inhibition. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive, may have important functions regulating gene expression and inhibition of ezrin activity by NSC305787 in osteosarcoma could be an attractive therapy to prevent clinically significant metastasis. Citation Format: Haydar Çelik, Gülay Bulut, Jenny Han, Garrett T. Graham, Tsion Z. Minas, Erin J. Conn, Sung-Hyeok Hong, Gary T. Pauly, Mutlu Hayran, Xin Li, Metin Özdemirli, Ayşe Ayhan, Michelle A. Rudek, Jeffrey A. Toretsky, Aykut Üren. Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2446.

Mode of Ezrin-Membrane Interaction as a Function of PIP2 Binding and Pseudophosphorylation

Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

Glypican 3 (GPC3), CD81 and HCV E2 protein control Yap and Hippo Pathway

Liver failure is a common outcome of end‐stage liver diseases. Modern stem cell and tissue engineering techniques can't achieve functional unique liver with multi cell types. Transplantation can only save limited amount of patients, the realistic approach for the majority of patients is to use residual functional liver tissue to restore impaired liver function. Maintaining dynamic balance, mitogenic and mitoinhibitory signals coexist in the human organs. This balance determines the fate of single cell, and determines the size and morphologic characterization of tissue or organ finally. Therefore, theoretically, restoration of functional liver mass in liver failure setting depends on either negative modulation of mitoinhibitory effect or positive modulation of mitogenic effect. One group of Japanese liver failure patients showed aberrantly up regulation of mitogenic signals such as HGF, indicates this blocked regeneration might be dominantly mediated by mitoinhibitory signals. Thus, the investigation of the mechanism of involving mitoinhibitory signaling may provide novel therapeutic targets for liver failure treatment.Previous studies from our laboratory have shown that GPC3 is associated with inhibition of hepatocyte growth and GPC3 transgenic mice with targeted expression in hepatocytes have decreased liver regeneration. Our previous investigation also showed that GPC3 overexpress intensively both in fulminant hepatic failure human liver tissues. We have also shown that GPC3 binds to the membrane tetraspanin known as CD81. Studies from other sources have shown that activation of CD81 is associated with highly targeted phosphorylation of the protein Ezrin. The latter has been shown to regulate the Hippo pathway, responsible for regulating nuclear levels of the protein known as Yes‐associated protein (Yap). Expression of Yap in the nucleus relates to enhanced liver size and activation of hepatocyte growth. Mice over‐expressing GPC3 with associated suppressed liver regeneration have decreased levels of nuclear Yap. We also found that treatment of primary rat hepatocytes with CD81 agonist antibody led to higher levels of phosphorylated Ezrin and lower Hippo activity, and this lower Hippo activity could be reversed by Spleen tyrosine kinase (Syk) inhibitor in highly differentiated hepatocytes. Partial hepatectomy (PHx) exhibited that loss of GPC3 antagonistic modulation during early liver regeneration, CD81 activated Ezrin and down regulated Hippo activity in vivo. Treatment with Hepatitis C virus (HCV) envelope protein E2 led to more Hippo activity and decrease in nuclear Yap in Hepa RG cells and primary human hepatocytes.ConclusionsDependent on Syk mediated CD81, GPC3 and HCV may function as growth inhibitory factor by modulating YAP expression pattern. The CD81‐Ezrin signaling enhances nuclear Yap levels. This pathway in normal hepatocytes is inhibited by GPC3. In human hepatocytes, GPC3 and the E2 protein of HCV also inhibit the CD81‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. Persistent HCV infection blocks the potential of liver regeneration by E2 protein interacting with CD81.Support or Funding InformationNIH/NCI R01‐CA103958

The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury

Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A alsostimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria wasgreater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

Abstract P5-01-01: Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer

Abstract Lymph node (LN) metastasis is a key driver of recurrence and survival in breast cancer (BC) patients. However, the mechanisms of metastatic dissemination of tumour cells from LNs to distant sites and their predictors of response to systemic therapy remain poorly understood, mainly due to a lack of non-invasive in vivo imaging models. We have recently described ezrin, a pro-metastatic crosslinker protein, as a regulator of tumour lymphangiogenesis and metastasis in BC (Breast Cancer Res. 2014; 16(5): 438). Furthermore, we demonstrated significant association of high ezrin expression with lymphovascular invasion in a cohort (n=63) of premenopausal patients with invasive BC (p =0.024). These findings prompted us to examine the role of ezrin in migration and invasion of metastatic tumour cells in LNs and their response to ezrin-targeted therapy. Using a locally accrued LN positive patient cohort (n=94), we demonstrated a significant association between high ezrin levels and reduced recurrence-free survival (univariate Log-rank test, p=0.033), suggesting that ezrin is a potential predictor of relapse in LN positive BC. To address the mechanistic role of ezrin in LN metastasis, we developed a novel intravital imaging model using a lymphatic reporter transgenic mouse (B6-prox1-mOrange2-pA-BAC) to examine the response of tumour-draining LN to anti-ezrin systemic therapy in real time. Next, we tested the effects of a small molecule ezrin inhibitor (NSC668394) in vitro and observed significant suppression of ezrin activation (p-T567) and cancer cell invasive phenotype. Intravital imaging of inguinal LN metastases, derived from subcutaneously implanted breast adenocarcinoma E0771-LMV (lung metastatic variant) cells, demonstrated significant reduction in mobility and invasiveness (Mann Whitney, p<0.0001) of metastatic cells following systemic treatment with NSC668394 (0.5 mg/kg at 24h and 8h prior to imaging). Interestingly, LN metastases engagement by host T cell (CD3+) was notably increased, whereas T cell mobility was not affected by ezrin inhibition. Our findings present a novel non-invasive imaging model to study the LN metastasis response to anti-cancer therapy in real time, and provide new insight into the role of ezrin as a potential anti-metastatic target in BC. (Supported by CRS, CIHR, CBCF, BCAK, Queen's SRC). Citation Format: Ghaffari A, Hoskin V, Mullins G, Greer P, Kiefer F, Madarnas Y, SenGupta S, Elliott B. Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-01.

Estrogen stimulated migration and invasion of estrogen receptor-negative breast cancer cells involves an ezrin-dependent crosstalk between G protein-coupled receptor 30 and estrogen receptor beta signaling

Estrogen mediates important cellular activities in estrogen receptor negative (ER-) breast cancer cells via membrane associated G protein-coupled receptor 30 (GPR30). However, the biological role and mechanism of estrogen action on cell motility and invasion in this aggressive kind of tumors remains poorly understood. We showed here that treatment with 17β-estradiol (E2) in ER-negative cancer cells resulted in ezrin-dependent cytoskeleton rearrangement and elicited a stimulatory effect on cell migration and invasion. Mechanistically, E2 induced ezrin activation was mediated by distinct mechanisms in different cell contexts. In SK-BR-3 cells with a high GPR30/ERβ ratio, silencing of GPR30 was able to abolish E2 induced ERK1/2, AKT phosphorylation and ezrin activation, whereas in MDA-MB-231 cells with low GPR30/ERβ ratio, E2 stimulated ezrin activation was mediated by the ERβ/PI3K/AKT signaling pathway. Importantly, we showed that activation of GPR30 signaling significantly prevents ERβ activation induced ezrin phosphorylation, cell migration and invasion, indicating an antagonist effect between GPR30 and ERβ signaling in MDA-MB-231 cells. These findings highlight the important interplay between different estrogen receptors in estrogen induced cell motility and invasiveness in ER-negative breast cancer cells.

CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering.

Cytoskeletal rearrangement is required for migration and invasion, which are the key steps of cancer metastasis. Ezrin and integrin co-ordinate these processes by regulating cellular adhesion and cytoskeletal polymerization-depolymerization. It is also well established that chemokine-chemokine receptor axis plays a crucial role in regulating cancer cell migration and invasion. In this study, we show involvement of CXC chemokine receptor 6 (CXCR6) and its only natural ligand CXCL16 in pathobiology of prostate cancer (PCa). CXCR6 is highly expressed in PCa tissues and cell lines (LNCaP and PC3), relative to normal tissue and cells. CXCR6 expression in PCa tissues correlated with higher Gleason score. Similarly, aggressive PCa cells (PC3) show high CXCR6 compared to less aggressive LNCaP. Besides, PC3 cells show higher MMPs expression compared to LNCaP cells following CXCL16 stimulation. Intriguingly, CXCR6-CXCL16 interaction in PCa cells promotes Ezrin activation, αvβ3 integrin clustering and capping at the leading edge in FAK/PI3K/PKC dependent manner, thereby modifying cellular adhesion as well as motility. Together these results demonstrate that CXCL16 stimulation changes cytoskeletal dynamics resulting in enhanced migration, invasion and adhesion to endothelial cells, ultimately enabling PCa cells to achieve their metastatic goal.

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization.

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

Abstract 3269: Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level

Abstract Ezrin is a member of the ezrin-radixin-moesin (ERM) family of actin-membrane linker proteins that play key roles in regulating cell shape, movement, adhesion and signal transduction pathways. The expression of ezrin is linked to the metastatic progression in several cancers including osteosarcoma (OS). We discovered a small molecule, NSC305787, that inhibits ezrin activity and metastatic phenotype both in vitro and in vivo. We hypothesized that the anti-metastatic effects of NSC308787 could be mediated through preventing specific protein-protein interactions involving ezrin. In this study, we used affinity pull-down coupled with mass spectrometry-based proteomic approach to unravel putative ezrin interactors that are competed away by NSC305787. We identified a number of candidate ezrin binding proteins that are associated with metastatic behavior and implicated in the regulation of stress granule dynamics and protein translation initiation. We selected DDX3, a DEAD-box RNA helicase, as a candidate for further analysis. We confirmed that ezrin directly binds to DDX3. Depletion of ezrin protein expression by RNA interference in several cancer cell lines resulted in substantial reduction in DDX3 protein levels without affecting its transcription, which suggested that ezrin is required for post-transcriptional maintenance of DDX3 in the cell. Paradoxically, recombinant ezrin specifically inhibited the RNA duplex unwinding activity and stimulated the ATPase activity of DDX3. Our data suggest that ezrin regulates the translation of mRNAs with 5′ secondary structure through DDX3, at least in part, through maintaining its intracellular protein level and/or modulating its unwinding and ATPase activities. Therefore, our findings suggest that a novel function of ezrin regarding the regulation of mRNA translation exists that is independent from its classical role as a cytoskeletal cross-linker protein at the plasma membrane. Citation Format: Haydar Celik, Kamal P. Sajwan, Amrita V. Pai, Ben J. Marsh, Yasemin Saygideger Kont, Said Rahim, Jenny Han, Tsion Minas, Jeffrey A. Toretsky, Aykut Uren. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3269. doi:10.1158/1538-7445.AM2015-3269

A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

Constitutively active ezrin desensitizes IL-7 signaling and impairs T cell homeostasis (LYM2P.735)

Abstract IL-7 signaling results in tethering of the IL-7 receptor to the actin cytoskeleton, which is mediated by the linker molecules ezrin and moesin. To understand the role of cytoskeleton linker molecules in cytokine signaling, we analyzed mice expressing a constitutively active ezrin transgene in T cells. The ezrin transgene was generated by introducing a point mutation at a conserved C-terminal Thr residue (567T->E) that results in a constitutively open conformation of ezrin. Interestingly, in mice expressing the constitutively active ezrin transgene (T567E-Tg), peripheral T cell numbers were significantly reduced and T cell survival was severely impaired. Moreover, T567E-Tg T cells were defect in IL-7 signaling even as they expressed high levels of surface IL-7 receptor proteins. Additionally, ezrin T567E-Tg induced downregulation of CD8 coreceptor expression, suggesting a “coreceptor tuning” effect of activated ezrin on CD8 T cells. Coreceptor tuning is a model of T cell homeostasis which posits that cytokine signals tailor CD8 coreceptor gene expression to the self-specificity of the TCR. In agreement, TCR disengagement restored IL-7 signaling in T567E-Tg CD8 T cells. Thus, constitutively active ezrin desensitizes IL-7 receptor signaling through increased TCR signaling but not through direct interference with the IL-7 receptor. The molecular mechanisms of ezrin activation on TCR signaling and T cell homeostasis will be discussed.

Slp2-a inactivates ezrin by recruiting protein phosphatase 1 to the plasma membrane

Synaptotagmin-like protein 2-a (Slp2-a) was originally described as a membrane trafficking protein that consists of a Slp homology domain (SHD), a linker domain, and tandem C2 domains (named the C2A domain and C2B domain). Slp2-a mediates docking of Rab27-bearing vesicles to the plasma membrane through simultaneous interaction with Rab27 and phospholipids in the plasma membrane. We have recently reported that Slp2-a regulates renal epithelial cell size through interaction with Rap1GAP2 via the C2B domain independently of Rab27 and demonstrated the presence of excess activation of ezrin, a membrane-cytoskeleton linker and signal transducer, in Slp2-a-knockdown Madin–Darby canine kidney II (MDCK II) cells. However, the precise mechanism of ezrin inactivation by Slp2-a in cell size control has remained largely unknown. In this study, we investigated the functional relationship between Slp2-a and ezrin in MDCK II cells. The results showed that activation of ezrin in control MDCK II cells either pharmacologically or by overexpression of a constitutively active ezrin mutant caused an increase in cell size, whereas inactivation of ezrin in Slp2-a-knockdown cells by a specific ezrin inhibitor restored them to their normal cell size. We also found that Slp2-a interacts via its previously uncharacterized linker domain with protein phosphatase 1β (PP1β), which inactivates ezrin, and that the interaction is required for the plasma membrane localization of PP1β. These results indicate that Slp2-a inactivates ezrin by recruiting PP1 to the plasma membrane.

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines.

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.