Tumor Biol. DOI 10.1007/s13277-014-2768-1
RESEARCH ARTICLE
CXCR7 signaling induced epithelial–mesenchymal transition by AKT and ERK pathways in epithelial ovarian carcinomas Hao Yu & Linlin Zhang & Peishu Liu
Received: 7 October 2014 / Accepted: 22 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Epithelial–mesenchymal transition (EMT) plays an important role in oncogenesis, through which cancer cells acquire an invasion and metastasis capacity. Notably, the chemokine receptor CXCR7 and its ligands CCL19 can also facilitate lymph node metastasis in epithelial ovarian carcinomas. Here, we assumed that CXCR7 might be involved in the EMT process of epithelial ovarian carcinomas. In our study, CXCR7 activation and inhibition in SKOV3 were induced with exogenous CCL19 and CXCR7 small interfering RNA (CXCR7 siRNA), respectively. AKT and ERK protein of CXCR7 pathways as well as biomarkers (vimentin, snail, Ncadherin, and E-cadherin) of EMT were detected using the Western blot. Our results showed that CCL19 can induce AKT and ERK phosphorylation in a dose-dependent fashion; however, CXCR7 siRNA efficaciously suppressed CCL19induced AKT and ERK phosphorylation in comparison with control siRNA. Importantly, CCL19 alone treatment can upregulate the expression of vimentin, snail, and N-cadherin of SKOV3 and downregulate the expression of E-cadherin. Conversely, knockdown of CXCR7 did not reveal any changes compared with CCL19 and the control. In conclusion, these findings demonstrate that EMT can be regulated by the
Hao Yu and Linlin Zhang contributed equally to this work. H. Yu : P. Liu (*) Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, No. 107 West Wenhua Road, Jinan 250012, Shandong, China e-mail: [email protected] H. Yu Department of Gynecologic Oncology, Shandong Cancer Hospital, Jinan 250117, Shandong, China L. Zhang Department of Obstetrics and Gynecology, Jinan Fourth People’s Hospital, Jinan, Shandong, China
CCL19/CXCR7 axis in epithelial ovarian carcinomas and then involved in the tumor cell invasion and metastasis process via activation of AKT and ERK pathways. Our study lays a new foundation for the treatment of epithelial ovarian carcinomas through antagonizing CXCR7. Keywords CXCR7 . EMT . AKT . ERK . Epithelial ovarian carcinomas
Introduction Epithelial ovarian carcinoma is the most common tumor of women and is the leading cause of gynecological cancerrelated deaths in women worldwide [1]. The dominative therapy involves surgery and platinum-based cytotoxic chemotherapy [2, 3]. This treatment can be curative for most patients with early-stage disease, but most women with advanced disease will develop many episodes of recurrent disease [4]. Therefore, understanding the molecular mechanisms of epithelial ovarian carcinoma is crucial to the treatment of ovarian cancer. Reportedly, the epithelial–mesenchymal transition has emerged as a key regulator in some cancers [5, 6]. With the development of epithelial–mesenchymal transition (EMT), epithelial cells lose their cell polarity and cell–cell adhesion and mesenchymal cells acquire migration and invasion capacity [7]. After EMT, epithelial cancer cells downregulate the expression of E-cadherin, but they promote N-cadherin, fibronectin, and vimentin expression. EMT is positively regulated by the transcription factors Snail and Twist that repress Ecadherin expression [8]. Several factors induce the EMT process, such as TGF-β and other growth factors that can activate the Wnt/β-catenin and Notch pathways to promote Snail and Slug expression [9, 10]. Based on all the above, the
Tumor Biol.
EMT pathway is of great importance in the treatment of cancer and could be targeted to prevent tumor dissemination [11, 12]. However, it is still unclear whether EMT is induced by chemokines and their receptors such as CCL19 and its receptor CXCR7. CXCR7 was demonstrated to play an essential role in cell migration and lymph node or distant metastasis of several types of cancer [13, 14]. Therefore, in the present study, we hypothesized that CXCR7 was involved in the EMT process of epithelial ovarian carcinomas.
Materials and methods Cell culture and reagents The human epithelial ovarian carcinoma cell line SKOV3 (serous papillary cystic adenocarcinoma) was obtained from the American Type Culture Collection (Manassas, VA). SKOV3 was maintained in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C. Recombinant human CCL19 was purchased from Sigma (St. Louis, USA). The CCL19 were then administered at gradient concentrations: 0 (the same concentration as that of PBS), 10, 20, 50, and 100 ng/ml. Antibodies were purchased from the same resources: antiCXCR7 antibody, anti-vimentin antibody, anti-Snail antibody, anti-N-cadherin antibody, anti-E-cadherin antibody, and anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Transfection of CXCR7 siRNA Cell lines were seeded in a 6-cm dish at a density of 5× 105 cells/dish and incubated overnight. SKOV3 was prepared for transfection of CXCR7 small interfering RNA (siRNA) or control siRNA (generously provided by Dr. Wang, Chinese Academy of Medical Sciences). siRNA was added to OptiMEM with Lipofectamine 2000 (Invitrogen) for transfection, according to the manufacturer’s instructions. Twelve hours following incubation, the medium was changed into fresh RPMI-1640 medium containing 10 % FBS. Cells were harvested at 72 h following transfection of CXCR7 siRNA. The transfection efficiency was assessed by flow cytometry. Efficiencies of CXCR7 siRNA and non-silencing control siRNA were tested using Western blot. The sequences of CXCR7 siRNA and control siRNA were the following: CXCR7 siRNA: Sense: 5′-GCGUCAACCCUUUCUUGUATT-3′ Anti-sense: 3′-UACAAGAAAGGGUUGACGCAG-5′
Control siRNA: Sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ Anti-sense: 3′-ACGUGACACGUUCGGAGAATT-5′
Western blotting Total protein from cell lines was extracted in cell lysis buffer (PIERCE, Rockford, IL) and quantified using the BSA method. A 10 % sodium dodecyl sulfate (SDS)–PAGE was performed, and 30 μg of protein of each sample was analyzed. Proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane by an electroblot apparatus. Membranes were incubated with primary antibodies. Antibody recognition was detected with either anti-mouse IgG or anti-rabbit IgG antibody linked to horseradish peroxidase (Sigma). Immunocomplexes were visualized by ECL (Amersham Pharmacia Biotech). Statistical analysis The data was presented as the mean±standard error of means (SEM). The analysis of difference was performed using oneway ANOVA with the least significant difference (LSD) post hoc test for multiple comparisons with SPSS version 13.0 software. The p
RESEARCH ARTICLE
CXCR7 signaling induced epithelial–mesenchymal transition by AKT and ERK pathways in epithelial ovarian carcinomas Hao Yu & Linlin Zhang & Peishu Liu
Received: 7 October 2014 / Accepted: 22 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Epithelial–mesenchymal transition (EMT) plays an important role in oncogenesis, through which cancer cells acquire an invasion and metastasis capacity. Notably, the chemokine receptor CXCR7 and its ligands CCL19 can also facilitate lymph node metastasis in epithelial ovarian carcinomas. Here, we assumed that CXCR7 might be involved in the EMT process of epithelial ovarian carcinomas. In our study, CXCR7 activation and inhibition in SKOV3 were induced with exogenous CCL19 and CXCR7 small interfering RNA (CXCR7 siRNA), respectively. AKT and ERK protein of CXCR7 pathways as well as biomarkers (vimentin, snail, Ncadherin, and E-cadherin) of EMT were detected using the Western blot. Our results showed that CCL19 can induce AKT and ERK phosphorylation in a dose-dependent fashion; however, CXCR7 siRNA efficaciously suppressed CCL19induced AKT and ERK phosphorylation in comparison with control siRNA. Importantly, CCL19 alone treatment can upregulate the expression of vimentin, snail, and N-cadherin of SKOV3 and downregulate the expression of E-cadherin. Conversely, knockdown of CXCR7 did not reveal any changes compared with CCL19 and the control. In conclusion, these findings demonstrate that EMT can be regulated by the
Hao Yu and Linlin Zhang contributed equally to this work. H. Yu : P. Liu (*) Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, No. 107 West Wenhua Road, Jinan 250012, Shandong, China e-mail: [email protected] H. Yu Department of Gynecologic Oncology, Shandong Cancer Hospital, Jinan 250117, Shandong, China L. Zhang Department of Obstetrics and Gynecology, Jinan Fourth People’s Hospital, Jinan, Shandong, China
CCL19/CXCR7 axis in epithelial ovarian carcinomas and then involved in the tumor cell invasion and metastasis process via activation of AKT and ERK pathways. Our study lays a new foundation for the treatment of epithelial ovarian carcinomas through antagonizing CXCR7. Keywords CXCR7 . EMT . AKT . ERK . Epithelial ovarian carcinomas
Introduction Epithelial ovarian carcinoma is the most common tumor of women and is the leading cause of gynecological cancerrelated deaths in women worldwide [1]. The dominative therapy involves surgery and platinum-based cytotoxic chemotherapy [2, 3]. This treatment can be curative for most patients with early-stage disease, but most women with advanced disease will develop many episodes of recurrent disease [4]. Therefore, understanding the molecular mechanisms of epithelial ovarian carcinoma is crucial to the treatment of ovarian cancer. Reportedly, the epithelial–mesenchymal transition has emerged as a key regulator in some cancers [5, 6]. With the development of epithelial–mesenchymal transition (EMT), epithelial cells lose their cell polarity and cell–cell adhesion and mesenchymal cells acquire migration and invasion capacity [7]. After EMT, epithelial cancer cells downregulate the expression of E-cadherin, but they promote N-cadherin, fibronectin, and vimentin expression. EMT is positively regulated by the transcription factors Snail and Twist that repress Ecadherin expression [8]. Several factors induce the EMT process, such as TGF-β and other growth factors that can activate the Wnt/β-catenin and Notch pathways to promote Snail and Slug expression [9, 10]. Based on all the above, the
Tumor Biol.
EMT pathway is of great importance in the treatment of cancer and could be targeted to prevent tumor dissemination [11, 12]. However, it is still unclear whether EMT is induced by chemokines and their receptors such as CCL19 and its receptor CXCR7. CXCR7 was demonstrated to play an essential role in cell migration and lymph node or distant metastasis of several types of cancer [13, 14]. Therefore, in the present study, we hypothesized that CXCR7 was involved in the EMT process of epithelial ovarian carcinomas.
Materials and methods Cell culture and reagents The human epithelial ovarian carcinoma cell line SKOV3 (serous papillary cystic adenocarcinoma) was obtained from the American Type Culture Collection (Manassas, VA). SKOV3 was maintained in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C. Recombinant human CCL19 was purchased from Sigma (St. Louis, USA). The CCL19 were then administered at gradient concentrations: 0 (the same concentration as that of PBS), 10, 20, 50, and 100 ng/ml. Antibodies were purchased from the same resources: antiCXCR7 antibody, anti-vimentin antibody, anti-Snail antibody, anti-N-cadherin antibody, anti-E-cadherin antibody, and anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Transfection of CXCR7 siRNA Cell lines were seeded in a 6-cm dish at a density of 5× 105 cells/dish and incubated overnight. SKOV3 was prepared for transfection of CXCR7 small interfering RNA (siRNA) or control siRNA (generously provided by Dr. Wang, Chinese Academy of Medical Sciences). siRNA was added to OptiMEM with Lipofectamine 2000 (Invitrogen) for transfection, according to the manufacturer’s instructions. Twelve hours following incubation, the medium was changed into fresh RPMI-1640 medium containing 10 % FBS. Cells were harvested at 72 h following transfection of CXCR7 siRNA. The transfection efficiency was assessed by flow cytometry. Efficiencies of CXCR7 siRNA and non-silencing control siRNA were tested using Western blot. The sequences of CXCR7 siRNA and control siRNA were the following: CXCR7 siRNA: Sense: 5′-GCGUCAACCCUUUCUUGUATT-3′ Anti-sense: 3′-UACAAGAAAGGGUUGACGCAG-5′
Control siRNA: Sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ Anti-sense: 3′-ACGUGACACGUUCGGAGAATT-5′
Western blotting Total protein from cell lines was extracted in cell lysis buffer (PIERCE, Rockford, IL) and quantified using the BSA method. A 10 % sodium dodecyl sulfate (SDS)–PAGE was performed, and 30 μg of protein of each sample was analyzed. Proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane by an electroblot apparatus. Membranes were incubated with primary antibodies. Antibody recognition was detected with either anti-mouse IgG or anti-rabbit IgG antibody linked to horseradish peroxidase (Sigma). Immunocomplexes were visualized by ECL (Amersham Pharmacia Biotech). Statistical analysis The data was presented as the mean±standard error of means (SEM). The analysis of difference was performed using oneway ANOVA with the least significant difference (LSD) post hoc test for multiple comparisons with SPSS version 13.0 software. The p
