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Nakagawa M, Matsumoto T, Yokoi A, Hashimura M, Oguri Y, Konno R, Ishibashi Y, Ito T, Ohhigata K, Harada Y, Fukagawa N, Kodera Y, Saegusa M. Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma. Mol Oncol 2023; 17:2168-2182. [PMID: 37539980 PMCID: PMC10552901 DOI: 10.1002/1878-0261.13503] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/29/2023] [Accepted: 08/01/2023] [Indexed: 08/05/2023] Open
Abstract
Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me-EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial-mesenchymal transition (EMT)-like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)-like properties. Shotgun proteomics analysis of proteins that co-immunoprecipitated with EBP50 revealed that Me-EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me-EBP50 KO, and blebbistatin treatment potentiated the effects of Me-EBP50 KO. In OCCC cells from clinical samples, Me-EBP50 and MYH9 were co-localized at the apical plasma membrane. Patients with a combination of Me-EBP50-high and MYH9-high scores had the best prognosis for overall and progression-free survival. Our data suggest that Me-EBP50 has tumor-suppressive effects through the establishment and maintenance of epithelial polarization. By contrast, loss of Me-EBP50 expression induces EMT-like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC-like properties, which in turn promote OCCC progression.
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Affiliation(s)
- Mayu Nakagawa
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Toshihide Matsumoto
- Department of PathologyKitasato University School of Allied Health ScienceSagamiharaJapan
| | - Ako Yokoi
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Miki Hashimura
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Yasuko Oguri
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Ryo Konno
- Center for Disease Proteomics, School of ScienceKitasato UniversitySagamiharaJapan
| | - Yu Ishibashi
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Takashi Ito
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Kensuke Ohhigata
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Yohei Harada
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Naomi Fukagawa
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
| | - Yoshio Kodera
- Center for Disease Proteomics, School of ScienceKitasato UniversitySagamiharaJapan
| | - Makoto Saegusa
- Department of PathologyKitasato University School of MedicineSagamiharaJapan
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Álvarez-Carrión L, Gutiérrez-Rojas I, Rodríguez-Ramos MR, Ardura JA, Alonso V. MINDIN Exerts Protumorigenic Actions on Primary Prostate Tumors via Downregulation of the Scaffold Protein NHERF-1. Cancers (Basel) 2021; 13:436. [PMID: 33498862 PMCID: PMC7865820 DOI: 10.3390/cancers13030436] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Revised: 01/19/2021] [Accepted: 01/20/2021] [Indexed: 11/17/2022] Open
Abstract
Advanced prostate cancer preferential metastasis to bone is associated with osteomimicry. MINDIN is a secreted matrix protein upregulated in prostate tumors that overexpresses bone-related genes during prostate cancer progression. Na+/H+ exchanger regulatory factor (NHERF-1) is a scaffold protein that has been involved both in tumor regulation and osteogenesis. We hypothesize that NHERF-1 modulation is a mechanism used by MINDIN to promote prostate cancer progression. We analyzed the expression of NHERF-1 and MINDIN in human prostate samples and in a premetastatic prostate cancer mouse model, based on the implantation of prostate adenocarcinoma TRAMP-C1 (transgenic adenocarcinoma of the mouse prostate) cells in immunocompetent C57BL/6 mice. The relationship between NHERF-1 and MINDIN and their effects on cell proliferation, migration, survival and osteomimicry were evaluated. Upregulation of MINDIN and downregulation of NHERF-1 expression were observed both in human prostate cancer samples and in the TRAMP-C1 model. MINDIN silencing restored NHERF-1 expression to control levels in the mouse model. Stimulation with MINDIN reduced NHERF-1 expression and triggered its mobilization from the plasma membrane to the cytoplasm in TRAMP-C1 cells. MINDIN-dependent downregulation of NHERF-1 promoted tumor cell migration and proliferation without affecting osteomimicry and adhesion. We propose that MINDIN downregulates NHERF-1 expression leading to promotion of processes involved in prostate cancer progression.
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Affiliation(s)
- Luis Álvarez-Carrión
- Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain; (L.Á.-C.); (I.G.-R.); (M.R.R.-R.)
| | - Irene Gutiérrez-Rojas
- Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain; (L.Á.-C.); (I.G.-R.); (M.R.R.-R.)
| | - María Rosario Rodríguez-Ramos
- Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain; (L.Á.-C.); (I.G.-R.); (M.R.R.-R.)
- Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain
| | - Juan A. Ardura
- Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain; (L.Á.-C.); (I.G.-R.); (M.R.R.-R.)
- Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain
| | - Verónica Alonso
- Bone Physiopathology Laboratory, Applied Molecular Medicine Institute (IMMA), Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain; (L.Á.-C.); (I.G.-R.); (M.R.R.-R.)
- Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, 28925 Alcorcón, Spain
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Wang Q, Song R, Zhao C, Liu H, Yang Y, Gu S, Feng D, He J. HPV16 E6 promotes cervical cancer cell migration and invasion by downregulation of NHERF1. Int J Cancer 2018; 144:1619-1632. [PMID: 30230542 DOI: 10.1002/ijc.31876] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Revised: 07/29/2018] [Accepted: 09/03/2018] [Indexed: 12/21/2022]
Abstract
HPV16 is the predominant type of HPV causing invasive cervical cancer. However, the underlying molecular mechanism of the unparalleled carcinogenic power of HPV16 compared to other types of high-risk (HR)-HPV including HPV18 remains elusive. The PDZ binding motif (PBM) of high-risk HPV E6 plays an important role in neoplasia and progression of cervical cancer. HPV16 E6 rather than HPV18 E6, interacted with NHERF1 by its PBM region, and induced degradation of NHERF1. NHERF1 retarded the assembly of cytoskeleton by downregulation of ACTN4, thereby inhibited the migration and invasion of cervical cancer cells in both cell and mouse model. HPV16 E6 was confirmed to enhance actin polymerization with increased ACTN4 level by downregulation of NHERF1, and result in enhanced migration and invasion of cervical cancer cells. GSEA analysis of cervical cancer specimens also showed that HPV16 E6 rather than HPV18 E6, was significantly associated with actin cytoskeleton assembly. That downregulation of NHERF1 by HPV16 E6 promoted cytoskeleton assembly and cell invasion, was an important cause in cervical cancer carcinogenesis. These findings provided the differential mechanism between HPV16 E6 and HPV18 E6 in the development and progression of cervical cancer, which may partially explain the differences of carcinogenic power between these two types of HR-HPVs.
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Affiliation(s)
- Qiqi Wang
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
| | - Ran Song
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
| | - Chunjuan Zhao
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
| | - Hua Liu
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
| | - Ying Yang
- Core Facilities Center, Capital Medical University, Beijing, People's Republic of China
| | - Siyu Gu
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
| | - Duiping Feng
- Department of Interventional Radiology, First Hospital of Shanxi Medical University, Taiyuan, People's Republic of China
| | - Junqi He
- Department of Biochemistry and Molecular Biology, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, People's Republic of China
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Phosphorylation of NHERF1 S279 and S301 differentially regulates breast cancer cell phenotype and metastatic organotropism. Biochim Biophys Acta Mol Basis Dis 2018; 1865:26-37. [PMID: 30326259 DOI: 10.1016/j.bbadis.2018.10.017] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Revised: 09/21/2018] [Accepted: 10/11/2018] [Indexed: 02/08/2023]
Abstract
Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.
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5
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Liu H, Zhao WL, Wang JP, Xin BM, Shao RG. EBP50 suppresses the proliferation of MCF-7 human breast cancer cells via promoting Beclin-1/p62-mediated lysosomal degradation of c-Myc. Acta Pharmacol Sin 2018; 39:1347-1358. [PMID: 29283175 DOI: 10.1038/aps.2017.171] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Accepted: 11/08/2017] [Indexed: 02/06/2023]
Abstract
c-Myc, a key activator of cell proliferation and angiogenesis, promotes the development and progression of breast cancer. Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is a multifunctional scaffold protein that suppresses the proliferation of breast cancer cells. In this study we investigated whether the cancer-suppressing effects of EBP50 resulted from its regulation of c-Myc signaling in human breast cancer MCF-7 cells in vitro and in vivo. We first found a significant correlation between EBP50 and c-Myc expression levels in breast cancer tissue, and demonstrated that EBP50 suppressed cell proliferation through decreasing the expression of c-Myc and its downstream proteins cyclin A, E and Cdc25A in MCF-7 cells. We further showed that EBP50 did not regulate c-Myc mRNA expression, but it promoted the degradation of c-Myc through the autophagic lysosomal pathway. Moreover, EBP50 promoted integration between c-Myc and p62, an autophagic cargo protein, triggering the autophagic lysosomal degradation of c-Myc. In EBP50-silenced MCF-7 cells, activation of autophagy by Beclin-1 promoted the degradation of c-Myc and inhibited cell proliferation. These results demonstrate that the EBP50/Beclin-1/p62/c-Myc signaling pathway plays a role in the proliferation in MCF-7 breast cancer cells: EBP50 stimulates the autophagic lysosomal degradation of c-Myc, thereby inhibits the proliferation of MCF-7 cells. Based on our results, promoting the lysosomal degradation of c-Myc might be a promising new strategy for treating breast cancer.
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Feng D, Xiong Y, Peng Z, Ma Q, Tao T, Liu H, Liang J, Wei Z, Zheng J, Wang L, Zhang H. Reduced EBP50 expression levels are correlated with unfavorable clinicopathological features of extrahepatic bile duct carcinoma and promote the proliferation and migration of QBC939 cells. Oncol Lett 2017; 13:2758-2764. [PMID: 28454463 DOI: 10.3892/ol.2017.5789] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2016] [Accepted: 10/27/2016] [Indexed: 12/21/2022] Open
Abstract
The present study aimed to clarify the association between ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) expression level and the tumor phenotype and clinicopathological features of extrahepatic bile duct carcinoma. Tissue samples from patients with extrahepatic bile duct carcinoma (54 cases) and patients with normal bile duct epithelia from gallbladder of cholecystitis (20 cases) were collected, and immunohistochemical staining was used to detect the expression levels of EBP50 in these tissues. In addition, small interfering (si)RNA-EBP50 was used to knock down the expression of EBP50 in the QBC939 human cholangiocarcinoma (CC) cell line. The effect of EBP50 expression on QBC939 cell proliferation and migration was analyzed using the Cell Counting kit-8 and wound healing assays, respectively. EBP50 expression was significantly downregulated in CC tissue samples (P<0.01), with low EBP50 expression levels positively correlated with a high pathological stage and a poor differentiation degree (P<0.01 and P<0.001, respectively). EBP50 expression in QBC939 cells was knocked down by ≤80% using siRNA-EBP50, and EBP50 knockdown significantly promoted QBC939 cell proliferation, as compared with the vector control cells (P=0.04). EBP50 knockdown also significantly enhanced the wound healing ability of QBC939 cells (P=0.02). These results demonstrated that EBP50 expression levels are significantly correlated with a malignant phenotype in patients with CC, and decreased expression levels of EBP50 may promote CC cell proliferation and migration. These findings provide insight into novel potential diagnostic and therapeutic approaches for patients with CC.
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Affiliation(s)
- Duiping Feng
- Department of Radiology, The First Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China.,Beijing Key Laboratory for Tumor Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, P.R. China
| | - Ying Xiong
- Beijing Key Laboratory for Tumor Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, P.R. China.,Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Zhiqiang Peng
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Qiang Ma
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Tao Tao
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Hua Liu
- Beijing Key Laboratory for Tumor Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, P.R. China.,Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Jianfang Liang
- Department of Pathology, The First Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Zhigang Wei
- Department of General Surgery, The First Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Junfang Zheng
- Beijing Key Laboratory for Tumor Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, P.R. China.,Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, P.R. China
| | - Lei Wang
- Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China
| | - Hui Zhang
- Department of Radiology, The First Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
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7
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Tao T, Yang X, Qin Q, Shi W, Wang Q, Yang Y, He J. NHERF1 Enhances Cisplatin Sensitivity in Human Cervical Cancer Cells. Int J Mol Sci 2017; 18:ijms18010005. [PMID: 28085111 PMCID: PMC5297640 DOI: 10.3390/ijms18010005] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2016] [Revised: 12/09/2016] [Accepted: 12/13/2016] [Indexed: 01/02/2023] Open
Abstract
Cervical cancer is one of the most common female malignancies, and cisplatin-based chemotherapy is routinely utilized in locally advanced cervical cancer patients. However, resistance has been the major limitation. In this study, we found that Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) was downregulated in cisplatin-resistant cells. Analysis based on a cervical cancer dataset from The Cancer Genome Atlas (TCGA) showed association of NHERF1 expression with disease-free survival of patients received cisplatin treatment. NHERF1 overexpression inhibited proliferation and enhanced apoptosis in cisplatin-resistant HeLa cells, whereas NHERF1 knockdown had inverse effects. While parental HeLa cells were more resistant to cisplatin after NHERF1 knockdown, NHERF1 overexpression in CaSki cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that NHERF1 significantly inhibited AKT and extracellular signal–regulated kinase (ERK) signaling pathways in cisplatin-resistant cells. Taken together, our results provide the first evidence that NHERF1 can sensitize cisplatin-refractory cervical cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumors.
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Affiliation(s)
- Tao Tao
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
| | - Xiaomei Yang
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
- Beijing International Cooperation Base for Science and Technology on China-UK Cancer Research, Beijing 100069, China.
| | - Qiong Qin
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
- Beijing International Cooperation Base for Science and Technology on China-UK Cancer Research, Beijing 100069, China.
| | - Wen Shi
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
- Experiment Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
| | - Qiqi Wang
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
| | - Ying Yang
- Core Facilities Center, Capital Medical University, Beijing 100069, China.
| | - Junqi He
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.
- Beijing International Cooperation Base for Science and Technology on China-UK Cancer Research, Beijing 100069, China.
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Zheng J, Wang L, Peng Z, Yang Y, Feng D, He J. Low level of PDZ domain containing 1 (PDZK1) predicts poor clinical outcome in patients with clear cell renal cell carcinoma. EBioMedicine 2016; 15:62-72. [PMID: 27993630 PMCID: PMC5233812 DOI: 10.1016/j.ebiom.2016.12.003] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Revised: 12/06/2016] [Accepted: 12/06/2016] [Indexed: 12/29/2022] Open
Abstract
Clear cell renal cell carcinoma (ccRCC) is the most lethal neoplasm of the urologic system. Clinical therapeutic effect varies greatly between individual ccRCC patients, so there is an urgent need to develop prognostic molecular biomarkers to help clinicians identify patients in need of early aggressive management. In this study, samples from primary ccRCC tumor and their corresponding nontumor adjacent tissues (n=18) were analyzed by quantitative proteomic assay. Proteins downregulated in tumors were studied by GO and KEGG pathways enrichment analyses. Six proteins were found both downregulated and annotated with cell proliferation in ccRCC patients. Of these proteins, PDZK1 and FABP1 were also involved in the lipid metabolism pathway. The downregulation of PDZK1 was further validated in TCGA_KIRC dataset (n=532) and independent set (n=202). PDZK1 could discriminate recurrence, metastasis and prognosis between ccRCC patients. Low level of PDZK1 in both mRNA and protein was associated with reduced overall survival (OS) and disease-free survival (DFS) in two independent sets. In univariate and multivariate analyses, PDZK1 was defined as an independent prognostic factor for both OS and DFS. These findings indicated that low level of PDZK1 could predict poor clinical outcome in patients with ccRCC.
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Affiliation(s)
- Junfang Zheng
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China; Beijing Key Laboratory for Tumor Invasion and Metastasis, Beijing International Cooperation Base for Science and Technology on China-UK Cancer Research, Beijing 100069, China
| | - Lei Wang
- Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
| | - Zhiqiang Peng
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China
| | - Ying Yang
- Core Facilities Center, Capital Medical University, Beijing 100069, China
| | - Duiping Feng
- Department of Radiology, First Hospital of Shanxi Medical University, Taiyuan 030001, China
| | - Junqi He
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China; Beijing Key Laboratory for Tumor Invasion and Metastasis, Beijing International Cooperation Base for Science and Technology on China-UK Cancer Research, Beijing 100069, China.
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9
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Liu H, Ma Y, He HW, Wang JP, Jiang JD, Shao RG. SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells. Autophagy 2016. [PMID: 26218645 DOI: 10.1080/15548627.2015.1074372] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
Abstract
Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.
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Affiliation(s)
- Hong Liu
- a Department of Oncology ; Institute of Medicinal Biotechnology; Peking Union Medical College; Chinese Academy of Medical Sciences ; Beijing , China
| | - Yan Ma
- a Department of Oncology ; Institute of Medicinal Biotechnology; Peking Union Medical College; Chinese Academy of Medical Sciences ; Beijing , China
| | - Hong-Wei He
- a Department of Oncology ; Institute of Medicinal Biotechnology; Peking Union Medical College; Chinese Academy of Medical Sciences ; Beijing , China
| | - Jia-Ping Wang
- b China Astronaut Research and Training Center ; Beijing , China
| | - Jian-Dong Jiang
- a Department of Oncology ; Institute of Medicinal Biotechnology; Peking Union Medical College; Chinese Academy of Medical Sciences ; Beijing , China
| | - Rong-Guang Shao
- a Department of Oncology ; Institute of Medicinal Biotechnology; Peking Union Medical College; Chinese Academy of Medical Sciences ; Beijing , China
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Millau JF, Wijchers P, Gaudreau L. High-Resolution 4C Reveals Rapid p53-Dependent Chromatin Reorganization of the CDKN1A Locus in Response to Stress. PLoS One 2016; 11:e0163885. [PMID: 27741251 PMCID: PMC5065170 DOI: 10.1371/journal.pone.0163885] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2016] [Accepted: 09/15/2016] [Indexed: 01/09/2023] Open
Abstract
A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stress-induced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from CDKN1A might act as a molecular switch to control spatial interactions between regulatory elements.
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Affiliation(s)
- Jean-François Millau
- Départment de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada
| | - Patrick Wijchers
- Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands
| | - Luc Gaudreau
- Départment de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada
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Peng Z, Wang Q, Zhang Y, He J, Zheng J. EBP50 interacts with EGFR and regulates EGFR signaling to affect the prognosis of cervical cancer patients. Int J Oncol 2016; 49:1737-45. [DOI: 10.3892/ijo.2016.3655] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 07/22/2016] [Indexed: 11/06/2022] Open
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12
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ZHENG JUNFANG, DAI YUANPING, YANG ZHIYU, YANG LONGYAN, PENG ZHIQIANG, MENG RAN, XIONG YING, HE JUNQI. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN. Oncol Rep 2015; 35:530-7. [DOI: 10.3892/or.2015.4375] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Accepted: 09/04/2015] [Indexed: 11/06/2022] Open
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Insulin-like growth factor and epidermal growth factor signaling in breast cancer cell growth: focus on endocrine resistant disease. Anal Cell Pathol (Amst) 2015; 2015:975495. [PMID: 26258011 PMCID: PMC4518167 DOI: 10.1155/2015/975495] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Accepted: 07/05/2015] [Indexed: 12/27/2022] Open
Abstract
Breast cancer is the most common type of cancer for women worldwide with a lifetime risk amounting to a staggering total of 10%. It is well established that the endogenous synthesis of insulin-like growth factor (IGF) and epidermal growth factor (EGF) polypeptide growth factors are closely correlated to malignant transformation and all the steps of the breast cancer metastatic cascade. Numerous studies have demonstrated that both estrogens and growth factors stimulate the proliferation of steroid-dependent tumor cells, and that the interaction between these signaling pathways occurs at several levels. Importantly, the majority of breast cancer cases are estrogen receptor- (ER-) positive which have a more favorable prognosis and pattern of recurrence with endocrine therapy being the backbone of treatment. Unfortunately, the majority of patients progress to endocrine therapy resistant disease (acquired resistance) whereas a proportion of patients may fail to respond to initial therapy (de novo resistance). The IGF-I and EGF downstream signaling pathways are closely involved in the process of progression to therapy resistant disease. Modifications in the bioavailability of these growth factors contribute critically to disease progression. In the present review therefore, we will discuss in depth how IGF and EGF signaling participate in breast cancer pathogenesis and progression to endocrine resistant disease.
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Yang L, Zheng J, Xiong Y, Meng R, Ma Q, Liu H, Shen H, Zheng S, Wang S, He J. Regulation of β2-adrenergic receptor cell surface expression by interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL). Amino Acids 2015; 47:1455-64. [PMID: 25876703 DOI: 10.1007/s00726-015-1965-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2014] [Accepted: 03/13/2015] [Indexed: 10/23/2022]
Abstract
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.
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Affiliation(s)
- Longyan Yang
- Department of Biochemistry and Molecular Biology, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing, 100069, People's Republic of China
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Li H, Zhang B, Liu Y, Yin C. EBP50 inhibits the migration and invasion of human breast cancer cells via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway. Med Oncol 2014; 31:162. [PMID: 25119502 DOI: 10.1007/s12032-014-0162-x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2014] [Accepted: 08/04/2014] [Indexed: 12/21/2022]
Abstract
The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50, also known as NHERF1 or NHERF) inhibits epidermal growth factor (EGF)-induced breast cancer cell proliferation after 3 days by blocking EGF receptor (EGFR) phosphorylation. The loss of EBP50 stimulates EGFR activity and induces the appearance of epithelial-to-mesenchymal transition phenotypic features in biliary cancer cells. However, the involvement of EBP50 in breast cancer migration and invasion remains unknown. We report that EBP50 inhibits the migration and invasion of breast cancer cells by inhibiting the phosphorylation of LIN-11, Isl1, and MEC-3 protein domain kinase, as well as cofilin. This phosphorylation is a critical step in cofilin recycling and actin polymerization mediating cytoskeletal rearrangement. Additionally, EGF-induced phosphorylation of Akt and mTOR was suppressed by upregulation of EBP50. Our results indicate that EBP50 is significantly involved in breast cancer invasion/metastasis via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway.
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Affiliation(s)
- Hongli Li
- Medicine Research Center, Weifang Medical University, Weifang, 261053, People's Republic of China
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Breast cancer-derived K172N, D301V mutations abolish Na+/H+exchanger regulatory factor 1 inhibition of platelet-derived growth factor receptor signaling. FEBS Lett 2013; 587:3289-95. [DOI: 10.1016/j.febslet.2013.08.026] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2013] [Revised: 08/12/2013] [Accepted: 08/26/2013] [Indexed: 11/21/2022]
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Lv XG, Lei XF, Ji MY, Guo XF, Wang J, Dong WG. Clinical significance of EBP50 overexpression assessed by quantum dot analysis in gastric cancer. Oncol Lett 2013; 5:1844-1848. [PMID: 23833653 PMCID: PMC3701077 DOI: 10.3892/ol.2013.1271] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Accepted: 03/01/2013] [Indexed: 12/15/2022] Open
Abstract
Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a postsynaptic density-95/disc-large/zonula occludens-1 (PDZ) homologous domain-containing protein that is involved in cell signaling. EBP50 regulates cell apoptosis, proliferation and invasion. In the present study, the prognostic impact factor of EBP50 expression was evaluated using a quantum dot (QD)-based assay and immunohistochemistry (IHC). The EBP50 protein expression in gastric cancer (GC) tissues was evaluated using IHC and QD-IHC. The study included 101 patients with GC (29 females and 72 males, aged 24-81 years), diagnosed and treated at the General Surgery Department of Renmin Hospital of Wuhan University (Wuhan, China) between 2000 and 2005. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. IHC and QD analyses of 101 GC tissue specimens revealed that EBP50-positive tumor cells were frequently present in GC. Increased EBP50 immunostaining was observed in 63 specimens (62.4%). The EBP50 expression levels were correlated with increased tumor size and the male gender. EBP50 was well distributed in the cytoplasm and nuclei of the GC cells. However, EBP50 protein expression exhibited no correlation with age, differentiation, stage or lymph node metastasis. There were no associations between the expression of EBP50 and the mean survival rates (IHC, 50.5 vs. 58.1 months, P>0.05; QD, 55.4 vs. 63.2 months, P>0.05). These findings suggest that EBP50 protein expression is not correlated with the prognosis of patients with GC. QD-IHC and IHC have similar advantages for the detection of EBP50 protein expression.
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Affiliation(s)
- Xiao-Guang Lv
- Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China
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Peng XL, Ji MY, Yang ZR, Song J, Dong WG. Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through β-catenin/E-cadherin pathway in human hepatocellular cancer. World J Gastroenterol 2013; 19:1306-1313. [PMID: 23483729 PMCID: PMC3587489 DOI: 10.3748/wjg.v19.i8.1306] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/09/2012] [Revised: 01/01/2013] [Accepted: 01/24/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) in hepatocellular carcinoma (HCC).
METHODS: Three human HCC cell lines, i.e., SM-MC7721, HepG2 and Hep3B, were used. We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50. Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells. In vitro cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution was assessed with flow cytometry. Invasion and migration ability of before and after the transfection were determined with a transwell assay. Cell apoptosis was demonstrated with Annexin V-FITC. The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.
RESULTS: The transfection efficiency was confirmed with Western blotting (1.36 ± 0.07 vs 0.81 ± 0.09, P < 0.01). The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells (P < 0.01). Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50 (61.3% ± 3.1% vs 54.0% ± 2.4%, P < 0.05). The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells (5.8 ± 0.8 vs 21.6 ± 1.3, P < 0.01). Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells (14.8% ± 2.7% vs 3.4% ± 1.3%, P < 0.05). The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells (0.28 ± 0.07 vs 0.56 ± 0.12, P < 0.05; 0.55 ± 0.08 vs 0.39 ± 0.07, P < 0.05). In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells (28.9 ± 7.2 vs 70.1 ± 7.2, P < 0.01).
CONCLUSION: The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin, E-cadherin. EBP50 may serve asa potential therapeutic target in HCC.
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Li X, Xu WM, Yin TL, Zhao QH, Peng LY, Yang J. Temporal and spatial regulation of ezrin-radixin-moesin-binding phosphoprotein-50-kDa (EBP50) during embryo implantation in mouse uterus. Int J Mol Sci 2012. [PMID: 23208378 PMCID: PMC3546698 DOI: 10.3390/ijms131216418] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Embryo implantation is a crucial process for successful pregnancy. To date, the mechanism of embryo implantation remains unclear. Ezrin-radixin-moesin-binding protein-50-kDa (EBP50) is a scaffold protein, which has been shown to play an important role in cancer development. Embryo implantation and cancer follow a similar progression. Thus, in this article, we utilized immunohistochemical staining and western blot analyses to examine the spatiotemporal expression and regulation of EBP50 both in the mouse uterus during embryo implantation as well as in other related models. We found that EBP50 was detected in epithelial cells in all of the groups used in our study. During the peri-implantation period, EBP50 mainly localized in apical membranes. At the implantation site (IS) on day 5 (D5) of pregnancy, EBP50 was mainly expressed in the nuclei of stroma cells, whereas from day 6 to day 8 (D6–D8) of pregnancy, the expression of EBP50 was noted in the cytoplasm of decidual cells. The expression of EBP50 was not significantly different in the pseudopregnant uterus and decreased in the uteri subjected to activation of delayed implantation. Artificial decidualization also decreased EBP50 expression. Thus, the expression levels and location were affected by active blastocysts and decidualization during the window of implantation.
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Affiliation(s)
- Xing Li
- Reproductive Medical Center, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei, China.
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Ji MY, Fan DK, Lv XG, Peng XL, Lei XF, Dong WG. The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells. J Mol Histol 2012; 43:517-26. [PMID: 22622406 DOI: 10.1007/s10735-012-9424-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Accepted: 04/29/2012] [Indexed: 12/19/2022]
Abstract
Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated β-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.
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Affiliation(s)
- Meng-Yao Ji
- Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei, China
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Cardone RA, Greco MR, Capulli M, Weinman EJ, Busco G, Bellizzi A, Casavola V, Antelmi E, Ambruosi B, Dell'Aquila ME, Paradiso A, Teti A, Rucci N, Reshkin SJ. NHERF1 acts as a molecular switch to program metastatic behavior and organotropism via its PDZ domains. Mol Biol Cell 2012; 23:2028-40. [PMID: 22496422 PMCID: PMC3364169 DOI: 10.1091/mbc.e11-11-0911] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Tumor metastasis is the primary cause of death in cancer patients, but the molecular mechanisms driving the evolution of the phenotype toward a specific organ is one of its less understood aspects. The scaffolding protein NHERF1 reprograms the metastatic phenotype and organotropism via the differential function of its PDZ domains. Metastatic cells are highly plastic for differential expression of tumor phenotype hallmarks and metastatic organotropism. The signaling proteins orchestrating the shift of one cell phenotype and organ pattern to another are little known. Na+/H+ exchanger regulatory factor (NHERF1) is a molecular pathway organizer, PDZ-domain protein that recruits membrane, cytoplasmic, and cytoskeletal signaling proteins into functional complexes. To gain insight into the role of NHERF1 in metastatic progression, we stably transfected a metastatic breast cell line, MDA-MB-231, with an empty vector, with wild-type NHERF1, or with NHERF1 mutated in either the PDZ1- or PDZ2-binding domains to block their binding activities. We observed that NHERF1 differentially regulates the expression of two phenotypic programs through its PDZ domains, and these programs form the mechanistic basis for metastatic organotropism. The PDZ2 domain promotes visceral metastases via increased invadopodia-dependent invasion and anchorage-independent growth, as well as by inhibition of apoptosis, whereas the PDZ1 domain promotes bone metastases by stimulating podosome nucleation, motility, neoangiogenesis, vasculogenic mimicry, and osteoclastogenesis in the absence of increased growth or invasion. Collectively, these findings identify NHERF1 as an important signaling nexus for coordinating cell structure with metastatic behavior and identifies the “mesenchymal-to-vasculogenic” phenotypic transition as an essential step in metastatic progression.
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Affiliation(s)
- Rosa Angela Cardone
- Department of Bioscience, Biotechnology and Pharmacological Sciences, University of Bari, Bari, Italy.
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EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation. Amino Acids 2012; 43:2027-35. [PMID: 22476347 PMCID: PMC3472071 DOI: 10.1007/s00726-012-1277-z] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2011] [Accepted: 03/14/2012] [Indexed: 12/12/2022]
Abstract
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.
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Ardura JA, Friedman PA. Regulation of G protein-coupled receptor function by Na+/H+ exchange regulatory factors. Pharmacol Rev 2011; 63:882-900. [PMID: 21873413 PMCID: PMC3186079 DOI: 10.1124/pr.110.004176] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Many G protein-coupled receptors (GPCR) exert patterns of cell-specific signaling and function. Mounting evidence now supports the view that cytoplasmic adapter proteins contribute critically to this behavior. Adapter proteins recognize highly conserved motifs such as those for Src homology 3 (SH3), phosphotyrosine-binding (PTB), and postsynaptic density 95/discs-large/zona occludens (PDZ) docking sequences in candidate GPCRs. Here we review the behavior of the Na+/H+ exchange regulatory factor (NHERF) family of PDZ adapter proteins on GPCR signalling, trafficking, and function. Structural determinants of NHERF proteins that allow them to recognize targeted GPCRs are considered. NHERF1 and NHERF2 are capable also of modifying the assembled complex of accessory proteins such as β-arrestins, which have been implicated in regulating GPCR signaling. In addition, NHERF1 and NHERF2 modulate GPCR signaling by altering the G protein to which the receptor binds or affect other regulatory proteins that affect GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the site of NHERF1-GPCR interaction are being developed and may become important and selective drug candidates.
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Affiliation(s)
- Juan A Ardura
- Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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Averna M, Stifanese R, De Tullio R, Minicucci L, Cresta F, Palena S, Salamino F, Pontremoli S, Melloni E. Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients. Biochim Biophys Acta Mol Basis Dis 2011; 1812:1649-57. [PMID: 21983488 DOI: 10.1016/j.bbadis.2011.09.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2011] [Revised: 09/21/2011] [Accepted: 09/23/2011] [Indexed: 02/04/2023]
Abstract
We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.
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Affiliation(s)
- Monica Averna
- Department of Experimental Medicine, University of Genoa, Viale Benedetto XV, Genoa, Italy
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Bartholow TL, Becich MJ, Chandran UR, Parwani AV. Immunohistochemical analysis of ezrin-radixin-moesin-binding phosphoprotein 50 in prostatic adenocarcinoma. BMC Urol 2011; 11:12. [PMID: 21672215 PMCID: PMC3132203 DOI: 10.1186/1471-2490-11-12] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2011] [Accepted: 06/14/2011] [Indexed: 12/19/2022] Open
Abstract
Background Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is an adapter protein which has been shown to play an active role in a wide variety of cellular processes, including interactions with proteins related to both tumor suppression and oncogenesis. Here we use immunohistochemistry to evaluate EBP50's expression in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). Methods Tissue microarrays were immunohistochemically stained for EBP50, with the staining intensities quantified using automated image analysis software. The data were statistically analyzed using one-way ANOVA with subsequent Tukey tests for multiple comparisons. Eleven cases of NDP, 37 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 103 cases of PCa, and 36 cases of Mets were analyzed in the microarrays. Results Specimens of PCa and Mets had the lowest absolute staining for EBP50. Mets staining was significantly lower than NDP (p = 0.027), BPH (p = 0.012), NAC (p < 0.001), HGPIN (p < 0.001), and PCa (p = 0.006). Additionally, HGPIN staining was significantly higher than NAC (p < 0.009) and PCa (p < 0.001). Conclusions To our knowledge, this represents the first study comparing the immunohistochemical profiles of EBP50 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC and suggests that EBP50 expression is decreased in Mets. Given that PCa also had significantly higher expression than Mets, future studies are warranted to assess EBP50's potential as a prognostic biomarker for prostate cancer.
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