Ran Meng

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.

NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability

The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na+/H+ exchanger regulatory factor (NHERF)‐1 loss‐of‐function and gain‐of‐function experiments reveal that polymerized actin cytoskeleton (F‐actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2‐dimensional electrophoresis‐matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α‐Actinin‐4, an actin cross‐linking protein, was identified. Glutathione S‐transferase pull‐down and coimmunoprecipitation studies showed the α‐actinin‐4 carboxyl‐terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc‐large/zona occludens‐1 domain. The NHERF1/α‐actinin‐4 interaction increased α‐actinin‐4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α‐actinin‐4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1.—Sun, L., Zheng, J., Wang, Q., Song, R., Liu, H., Meng, R., Tao T., Si, Y., Jiang, W., He, J. NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability. FASEB J. 30, 578‐589 (2016). www.fasebj.org

MAGI3 Suppresses Glioma Cell Proliferation via Upregulation of PTEN Expression

OBJECTIVEnTo investigate the role and molecular mechanism of membrane-associated guanylate kinase inverted 3 (MAGI3) in glioma cell proliferation.nnnMETHODSnThe expression levels of MAGI3 and PTEN were assessed in glioma samples by Western blotting. MAGI3 was stably transfected into C6 glioma cells to obtain C6-MAGI3 cells. Then, the proliferation, the expression levels of MAGI3 and PTEN, and Akt phosphorylation were evaluated in C6 and C6-MAGI3 cells. Xenograft tumor models were established by subcutaneous injection of C6 and C6-MAGI3 cells into nude mice, and the growth rates of xenografts in the mice were compared. The potential role of MAGI3 expression in PI3K/Akt signaling activation was further investigated by examining the correlation between MAGI3 expression and the expression of PI3K/Akt signaling downstream target genes in a glioma dataset using gene set enrichment analysis (GSEA).nnnRESULTSnExpression levels of MAGI3 and PTEN were significantly downregulated in gliomas. Overexpression of MAGI3 in the glioma C6 cell line upregulated PTEN protein expression, inhibited the phosphorylation of Akt, and suppressed cell proliferation. MAGI3 overexpression also inhibited the growth of C6 glioma tumor xenografts in nude mice. Analysis based on the GEO database confirmed the negative correlation between activation of PI3K/Akt pathway and MAGI3 mRNA levels in human glioma samples.nnnCONCLUSIONnThe loss of MAGI3 expression in glioma may enhance the proliferation of glioma cells via downregulation of PTEN expression, leading to the activation of the PI3K/Akt pathway. MAGI3 is a potential glioma suppressor.

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.

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.

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.

NHERF1 inhibits proliferation of triple-negative breast cancer cells by suppressing GPER signaling

G protein-coupled estrogen receptor (GPER) signaling is activated in triple-negative breast cancer (TNBC); however, the detailed mechanisms of its regulation remain unclear. The present study aimed to elucidate the molecular mechanisms involved in GPER activation in TNBC. In MDA-MB-231 cells, a TNBC cell line, NHERF1 interaction with GPER was verified by co-immunoprecipitation and immunofluorescent staining assays. Overexpression of NHERF1 in MDA-MB-231 cells inhibited GPER-mediated proliferation and phosphorylation of ERK1/2 and Akt. Furthermore, NHERF1 expression levels were negatively correlated with the gene signatures of GPER activation, ERK1/2 and Akt signaling, and cell proliferation in early stage of TNBC tumors from the TCGA data set. Taken together, NHERF1 inhibited the activation of GPER-mediated signaling and suppressed the proliferation of triple-negative breast cancer cells. Loss of NHERF1 expression may play a pivotal role in the early stage of TNBC carcinogenesis.

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 1149: Interaction with NHERF1 enhances protein stability of G protein-coupled estrogen receptor

G Protein-Coupled Estrogen Receptor (GPER), also called GPR30, has been demonstrated to play important roles in a variety of physiological and pathological responses, especially in cancer cells proliferation, migration and invasion. Despite increasing evidences have showed clinical correlations between GPER expression level and poor prognosis in human cancer specimens, little is known regarding the regulation of GPER expression level, especially in protein level. Our previous studies have shown that PDZ protein NHERF1 can interact with ACTN4 (FASEB J. 2015) or PTEN (Endocrinology. 2011) and prevent their degradation. In this study, NHERF1 was demonstrated to interact with GPER in HEK293 cells and breast cancer cells MCF7 and T47D. GST Pull-down, Co-IP and immunocytochemistry analysis revealed that this interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal of GPER. Mutation of the last c-terminal amino acid of GPER V375A could abolish the interaction of NHERF1 with GPER. Change of NHERF1 expression level by overexpression or knockdown affects the overall protein level of GPER. NHERF1 overexpression improved the GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in an interaction dependent manner. Further analysis of public TCGA data revealed that NHERF1 expression level positively associated with activation of GPER downstream genes in invasive breast cancer, in which GPER mRNA level was similar with that of normal breast tissue. Our findings identify NHERF1 as a novel binding partner for GPER and uncover a novel role of NHERF1 in promoting GPER protein stability by inhibiting ubiquitin-proteasome degradation of the receptor. Considering the importance of GPER in breast cancer progression, these findings may help elucidate mechanisms associated with breast cancer invasion and metastasis. Citation Format: Ran Meng, Ying Xiong, Yuan Zhao, Yan Wang, Tao Tao, Qiqi Wang, Hua Liu, Songlin Wang, Qiong Qin, Junfang Zheng, Junqi He. Interaction with NHERF1 enhances protein stability of G protein-coupled estrogen receptor. [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 1149.