Longyan Yang

Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) Is Required for the Estradiol-Dependent Increase of Phosphatase and Tensin Homolog (PTEN) Protein Expression

Expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) can be induced by estrogens at the posttranscriptional level. However, the molecular mechanism of the process is unclear. In this study, we found that the C terminus (CT) of PTEN is indispensable for 17-β-estradiol (E2)-increased PTEN expression. Therefore, we screened for PTEN-CT-associated proteins using a glutathione-S-transferase pull-down approach in combination with mass spectrometry-based proteomic analyses. Our experiments led to the identification of Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) as a major PTEN-CT binding partner. The first postsynaptic density protein-95/Discslarge/zonula occludens-1 homology domain of NHERF1 and the last four amino acids of PTEN were found to be key determinants of this interaction. By associating with PTEN, NHERF1 could enhance PTEN protein expression by retention of PTEN turnover, as demonstrated by NHERF1 overexpression and small interfering RNA-mediated knockdown experiments, respectively. Furthermore, NHERF1 inhibited ubiquitination of the PTEN protein upon competition with binding of PTEN to neural precursor cell expressed, developmentally down-regulated 4, an ubiquitin E3 ligase. E2 strongly induced the expression of NHERF1 and PTEN only in estrogen receptor (ER)-positive cells but not in ER-negative cells. ICI182780, an ER-specific inhibitor, decreased the expression of both NHERF1 and PTEN, and ICI182780 pretreatment also retarded E2-increased PTEN expression in ER-MDA-MB-231 cells. In both ER-MDA-MB-231 and MCF-7 cells, E2 failed to increase PTEN expression when NHERF1 was knocked down. Taken together, these are the first results that present a possible mechanism for E2-increased PTEN expression. In this process, E2 first induces NHERF1 expression by activating the ER. Upon competition with neural precursor cell expressed, developmentally down-regulated 4, NHERF1 then interacts with PTEN to inhibit PTEN degradation, through an ubiquitination-dependent pathway. This in turn leads to the increase of PTEN expression at the protein level.

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

MAGI3 negatively regulates Wnt/β-catenin signaling and suppresses malignant phenotypes of glioma cells.

Gliomas are the most common primary brain malignancies and are associated with a poor prognosis. Here, we showed that the PDZ domain-containing protein membrane-associated guanylate kinase inverted 3 (MAGI3) was downregulated at the both mRNA and protein levels in human glioma samples. MAGI3 inhibited proliferation, migration, and cell cycle progression of glioma cells in its overexpression and knockdown studies. By using GST pull-down and co-immunoprecipitation assays, we found that MAGI3 bound to β-catenin through its PDZ domains and the PDZ-binding motif of β-catenin. MAGI3 overexpression inhibited β-catenin transcriptional activity via its interaction with β-catenin. Consistently, MAGI3 overexpression in glioma cells C6 suppressed expression of β-catenin target genes including Cyclin D1 and Axin2, whereas MAGI3 knockdown in glioma cells U373 and LN229 enhanced their expression. MAGI3 overexpression decreased growth of C6 subcutaneous tumors in mice, and inhibited expression of β-catenin target genes in xenograft tumors. Furthermore, analysis based on the Gene Expression Omnibus (GEO) glioma dataset showed association of MAGI3 expression with overall survival and tumor grade. Finally, we demonstrated negative correlation between MAGI3 expression and activity of Wnt/β-catenin signaling through GSEA of three public glioma datasets and immunohistochemical staining of clinical glioma samples. Taken together, these results identify MAGI3 as a novel tumor suppressor and provide insight into the pathogenesis of glioma.

Breast cancer‐derived K172N, D301V mutations abolish Na+/H+ exchanger regulatory factor 1 inhibition of platelet‐derived growth factor receptor signaling

Na+/H+ exchanger regulatory factor 1 (NHERF1) is a scaffold protein known to interact with a number of cancer‐related proteins. nherf1 Mutations (K172N and D301V) were recently identified in breast cancer cells. To investigate the functional properties of NHERF1, wild‐type and cancer‐derived nherf1 mutations were stably expressed in SKMES‐1 cells respectively. NHERF1‐wt overexpression suppressed the cellular malignant phenotypes, including proliferation, migration, and invasion. nherf1 Mutations (K172N and D301V) caused complete or partial loss of NHERF1 functions by affecting the PTEN/NHERF1/PDGFRβ complex formation, inactivating NHERF1 inhibition of PDGF‐induced AKT and ERK activation, and attenuating the tumor‐suppressor effects of NHERF1‐wt. These results further demonstrated the functional consequences of breast cancer‐derived nherf1 mutations (K172N and D301V), and suggested the causal role of NHERF1 in tumor development and progression.

Ang-(1-7) promotes the migration and invasion of human renal cell carcinoma cells via Mas-mediated AKT signaling pathway.

Ang-(1-7) is an active peptide component of renin-angiotensin system and endogenous ligand for Mas receptor. In the current study, we showed that Ang-(1-7) enhanced migratory and invasive abilities of renal cell carcinoma cells 786-O and Caki-1 by wound-healing, transwell migration and transwell invasion assays. Mas antagonist A779 pretreatment or shRNA-mediated Mas knockdown abolished the stimulatory effect of Ang-(1-7). Furthermore, Ang-(1-7)-stimulated AKT activation was inhibited by either A779 pretreatment or Mas knockdown. Blockage of AKT signaling by AKT inhibitor VIII inhibited Ang-(1-7)-induced migration and invasion in 786-O cells. Taken together, our results provided the first evidence for the pro-metastatic role of Ang-(1-7) in RCC, which may help to better understand the molecular mechanism underlying the progression of this tumor.

Stabilization of the angiotensin-(1–7) receptor Mas through interaction with PSD95

The functions and signalling mechanisms of the Ang-(1-7) [angiotensin-(1-7)] receptor Mas have been studied extensively. However, less attention has been paid to the intracellular regulation of Mas protein. In the present study, PSD95 (postsynaptic density 95), a novel binding protein of Mas receptor, was identified, and their association was characterized further. Mas specifically interacts with PDZ1-2, but not the PDZ3, domain of PSD95 via Mas-CT (Mas C-terminus), and the last four amino acids [ETVV (Glu-Thr-Val-Val)] of Mas-CT were determined to be essential for this interaction, as shown by GST pull-down, co-immunoprecipitation and confocal co-localization experiments. Gain-of-function and loss-of-function studies indicated that PSD95 enhanced Mas protein expression by increasing the stabilization of the receptor. Mas degradation was robustly inhibited by the proteasome inhibitor MG132 in time- and dose-dependent manners, and the expression of PSD95 impaired Mas ubiquitination, indicating that the PSD95-Mas association inhibits Mas receptor degradation via the ubiquitin-proteasome proteolytic pathway. These findings reveal a novel mechanism of Mas receptor regulation by which its expression is modulated at the post-translational level by ubiquitination, and clarify the role of PSD95, which binds directly to Mas, blocking the ubiquitination and subsequent degradation of the receptor via the ubiquitin-proteasome proteolytic pathway.

Regulation of β2-adrenergic receptor cell surface expression by interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL)

The beta-2 adrenergic receptor (β2AR), a member of GPCR, can activate multiple signaling pathways and is an important treatment target for cardiac failure. However, the molecular mechanism about β2AR signaling regulation is not fully understood. In this study, we found that cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) overexpression reduced β2AR-mediated extracellular signal-regulated kinase-1/2 (ERK1/2) activation. Further study identified CAL as a novel binding partner of β2AR. CAL is associated with β2AR mainly via the third intracellular loop (ICL3) of receptor and the coiled-coil domains of CAL, which is distinct from CAL/β1AR interaction mediated by the carboxyl terminal (CT) of β1AR and PDZ domain of CAL. CAL overexpression retarded β2AR expression in Golgi apparatus and reduced the receptor expression in plasma membrane.

Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular target for antineoplastic therapy.

Abstract 1138: Breast cancer-derived mutations abolish Na+/H+ exchanger regulatory factor 1 inhibition of platelet-derived growth factor signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Na+/H+ exchanger regulatory factor 1 (NHERF1, also known as EBP50) is a scaffold protein known to interact with a number of target proteins, including cancer-related proteins such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), neurofibromatosis 2, spleen tyrosine kinase, platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR) and β-catenin. This suggests a possible function of NHERF1 as a negative tumor regulator. Several lines of evidence have suggested that NHERF1 is involved in carcinogenesis. However, the mechanisms by which NHERF1 plays a role in breast cancer have not yet been clearly defined. Three point mutations in nherf1 (K172N, R180W, and D301V) were recently identified in breast cancer cell lines or primary breast tumors. To gain more insight into the functional properties of NHERF1, wild-type (wt) and cancer-derived nherf1 mutations were introduced into a human non-small cell lung cancer cell line, SKMES-1. NHERF1-wt overexpression suppressed the malignant phenotypic characteristics of SKMES-1 cells, including cell proliferation, migration, and invasion. Three nherf1 mutations led to complete or partial loss of NHERF1 functions by affecting the formation of PTEN/NHERF1/PDGFRβ complex, attenuating NHERF1-mediated inhibition of PDGF-induced AKT and ERK activation, and abolishing the tumor suppressor effects mediated by NHERF1-wt. These results provide the first evidence of the functional consequences and signaling properties of breast cancer-derived nherf1 mutations, and suggest that these nherf1 mutations might play a causal role in tumor development and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2011-1138

Abstract 1935: EBP50 inhibits breast cancer cell proliferation by blocking EGFR phosphorylation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL EBP50 (Ezrin-radixin-moesin-binding phosphoprotein-50, also known as NHERF, NHERF1) has been reported to interact with epidermal growth factor receptor (EGFR) and enhance EGFR activity and its downstream ERK signaling in a cervical cancer cell line HeLa. However, our previous results demonstrated that EBP50 could suppress breast cancer cell proliferation, suggesting its inhibitory effect on EGFR signaling in breast cancer cell. So we detected the effect of EBP50 expression on EGF-induced cell proliferation and the activation of EGFR signaling in breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells which expressed low level of EBP50, EBP50 over-expression inhibited EGF-induced cell proliferation. In MCF-7 cells which expressed high level of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation. EBP50 was reported to interact with EGFR via its carboxyl terminal regulatory domain, which is adjacent to the autophosphorylation domain of the receptor, so it is possible that the association of EBP50 and EGFR mask the phosphorylation site of EGFR so as to prevent EGFR activation. To test this hypothesis, we detected the phosphorylation status of EGFR in the presence or absence of the EBP50 expression. Over-expression of EBP50 in MDA-MB-231 inhibits EGF-stimulated EGFR phosphorylation, and knockdown of EBP50 in MCF-7 cells led to the enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, the total expression levels of EGFR were not affected by the different expression level of EBP50 during EGF stimulation. In the case of EBP50 expression on the EGFR downstream signaling, EBP50 over-expression inhibited EGF-stimulated ERK activity in MDA-MB-231 cells, and EBP50 knockdown resulted in the enhanced EGF-stimulated ERK activity in MCF-7 cells. These data are consistent with results of MDA-MB-231 and MCF-7 cell lines pretreated with EGFR tyrosine kinase inhibitor-AG1478, that EGF-stimulated ERK activity was inhibited when EGFR phosphorylation was blocked. Taken together, our results demonstrated that EBP50 can suppress EGF-induced proliferation of breast cancer cells by retarding EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insights into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1935. doi:10.1158/1538-7445.AM2011-1935