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Cellular prion protein regulates invasion and migration of breast cancer cells through MMP-9 activity
Biochemical and Biophysical Research Communications 470 (2016) 213e219
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cellular prion protein regulates invasion and migration of breast cancer cells through MMP-9 activity Minchan Gil a, Yun Kye Kim d, Kyung-Eun Kim a, Wook Kim c, Chan-Sik Park b, **, Kyung Jin Lee d, * a
SIS Immunology Research Institute, Sookmyung Women's University, Seoul, South Korea Department of Pathology, Cell Dysfunction Research Center, University of Ulsan College of Medicine, Seoul, South Korea Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea d Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 138-736, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2015 Accepted 7 January 2016 Available online 9 January 2016
Function of cellular prion protein (PrPc) in cancer progression has not been elucidated yet. Ectopic expression of PrPc increases the invasion and migration of breast cancer cell line, MCF-7 cells. Overexpressed PrPc increases matrix metalloprotease-9 (MMP-9) expression by enhancing association of NF-kB in promoter of MMP-9 gene and ERK signaling in MCF-7 cells. Whereas, silencing of PrPc by siRNA suppresses ERK activation and MMP-9 expression resulting the down-regulation of MD-MB231 cell migration and invasion. Overall, these results suggest that PrPc contribute the breast cancer invasion and migration via MMP-9. © 2016 Elsevier Inc. All rights reserved.
Keywords: Prion Matrix metalloprotease-9 Migration Invasion Breast cancer
1. Introduction Cellular prion protein (PrPc) is a glycosylphosphatidylinositollinked extracellular membrane glycoprotein, which has been extensively studied for its critical role in the pathogenesis of transmissible spongiform encephalopathies. A pathogenic conformational isoform PrPSc is an infectious agent in Prion disease [1e3]. PrPc is highly expressed in neurons along with wild range of expression in various tissues. Physiological function of normal PrPc expression has been investigated in various cell types has been shown to be involved in many important biological processes, such as cell adhesion, migration, proliferation, neuroprotection, and inflammation [4e6]. However, the precise physiological role of PrPc remains incompletely understood in various types of cancer cell. PrPc is highly expressed in various cancer cells including glioma [7], breast cancer [8,9], prostate tumor [10], gastric cancer [11], colorectal cancer [12], pancreatic cancer [13], and osteosarcoma
Abbreviations: AP-1, activator protein-1; Ab, antibody; PrPc, cellular prion protein; MMP-9, matrix metalloprotease-9. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.-S. Park), [email protected] (K.J. Lee). http://dx.doi.org/10.1016/j.bbrc.2016.01.038 0006-291X/© 2016 Elsevier Inc. All rights reserved.
[14]. In breast cancer, higher expressions of PrPc are associated with a poor prognosis [15]. The role of PrPc to breast cancer cells has been investigated in several studies [8,9,16e18]. Overexpression of PrPc protects the breast cancer cell from TNF-a induced cell death by preventing Bax-mediated cytochrome C release [8,16]. Silencing of PrPc sensitizes Adriamycin-resistance carcinoma cells to TRAILmediated cell death [9]. Additionally, PrPc mediates the Paclitaxel-induced invasion of breast cancer cells [17]. By contrast, knockdown of PrPc in breast cancer cells are more resistance to doxorubicin treatment [18]. However, it remains unclear whether PrPc contributes to metastasis of breast cancer cells. Metastasis is composed of complex, multi-step process that involves the separation of tumor cells from original tumor site, intravasation, extravasation, and the settlement of tumor cells at a secondary site [19]. The invasiveness of tumor cells is critical in the all metastasis processes. Several proteases, including matrix metalloproteinase's (MMPs), cysteine proteinases, and serine proteases, are required for invasion because invasion requires altering extracellular matrix to permit the egress and ingress of cancer cells [20e22]. Among the MMPs, MMP-9 are thought to be directly involved in cancer cell growth and progression during metastasis [23,24]. Increased MMP-9 activity is observed in various tumor cells such as human breast cancer, lung cancer and ovarian cancer [25,26].
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In this study, we investigated the role of PrPc on invasion and migration of breast cancer cell lines by overexpression and knockdown of PrPc. Our results suggest that PrPc increase the migration and invasion in of breast cancer by activating NF-kB and ERK pathway. 2. Materials and methods 2.1. Establishment of prion-overexpressing stable transfectants PrPc overexpressing plasmids (kindly provided by Dr. Greene LE) were transfected into MCF-7 and MDA-MB231 (American Type Culture Collection, Manassas, VA) cells with lifopectamine 2000 (Invitrogen, Eugene, OR) and subjected to antibiotic selection for 1 month. The stable transfectant with highest PrPc expression were selected for further experiments. 2.2. RNA preparation, semi-quantitative RT-PCR Total RNA was extracted from the cells using NucleoSpin (Macherey-Nagel, Düren, Germany). The PCR amplification protocol was 30 cycles of 94 C for 30 s, 56 C for 30 s, and 72 C for 1 min. Amplified products were resolved by 1.5% agarose gel electrophoresis. The primers that were used are 50 -GGCAGTGACTATGAGGA eCCGTTAC-30 and 50 -GGCTTGACCAGCATCTCAGGTCTA-30 for human prion; 50 -TGACAGCGACA- AGAAGTG-30 and 50 -CAGTGAAGCGGTACATAGG-30 for MMP-9; 50 -CAATTCCGACCTCGTCATCA-30 and 50 -TCAGAGCCTTGGAGGAGCT-30 for TIMP-1 50 GATGATATCGC- CGCGCTCGTCGTCGAC-30 and 50 -GCCAGGTCCAGACGCAGGATGGCATG-30 and 50 AGCCAGGTCCA0 GACGCAGGATGGCATG-3 for b-actin. 2.3. Western blot analysis Cells were harvested and washed 3 times with cold phosphatebuffered saline (PBS). The cytoplasmic and nuclear protein fractions were extracted using NE-PER extraction reagents according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL). Nuclear protein extracts or whole protein extracts were used for Western blot analysis. Western blotting was performed using antiprion, anti-MMP-9, anti-JNK/ERK/p38, anti-phospho-JNK/ERK/p38, anti-Lamine and anti-actin antibodies. Protein samples were heated at 95 C for 5 min and were analyzed using SDSePAGE. Immunoblot signals were developed by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA).
2.5. Cell migration assay MCF-7 cells were seeded on collagen-covered 35-mm plates at a density of 0.4 106 per plate. After 24 h, the medium was replaced by 0.5% FBS containing DMEM, in each plate; three areas were scratched, creating three gaps of similar widths. Immediately thereafter, and at 24 h later, phase-contrast images of the plates were obtained with a CCD camera connected to an Olympus fluorescent microscope (X 10 objective). The region imaged at zero time was marked to enable us to photograph the same area at different times, thus allowing examination of a specific population of migrating cells. The widths of gaps were measured by Image-Pro Plus software. The data acquired from the three scratches on each plate were averaged to obtain the mean gap width.
2.6. Gelantin zymography MMP-9 enzymatic activity was accessed by gelatin zymography. The culture media were subjected to non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 0.2% gelatin-containing gels. After electrophoresis, gels were washed in 2.5% Triton X-100 to remove the SDS; incubated for 22 h at 37 C in 50 mm TriseHCl, pH 7.5, containing 0.15 M NaCl, 10 mm CaCl2, and 0.02% NaN3; and stained with 0.1% coomassie brilliant blue R250.
2.7. Transient transfection and luciferase assay The luciferase assays were performed using the luciferase assay system and the b-galactosidase assay system (Promega, Madison, WI). Transfection of MCF-7 cells was performed using Lipofectamine 2000 (Invitrogen). A standard transfection reaction used 1 mg of DNA, including the MMP-9 proximal promoter reporter constructs (kindly gifted from Dr. Wolfgang Eberhardt), internal control construct (pM1-b-gal), and expression construct (pCDNA3.1 or pCDNA3.1-prion). Luciferase activities were normalized with respect to b-galactosidase activities. The data shown are a representative of three independent experiments.
2.8. Chromatin immunoprecipitation Chromatin immunoprecipitation was carried out with Chromatin Immunoprecipitation Assay Kit (Upstate Biotechnology Inc., Lake Placid, NY) according to the manufacture's protocol.
2.9. RNA interference 2.4. Cell invasion assay Invasion assay was performed using a Transwell chamber (Costar, Cambridge, MA, USA) with 8 mm pore polycarbonate filters precoated with Matrigel (BD Biosciences, Franlin Lakes, NJ, USA). Briefly, 5 104 cells were harvested and suspended in 100 ml serum-free media and added to the upper chambers of the transwell system. Medium containing 10% FBS was placed into the lower chamber. After 24 h, non-migrated cells and media on the upper chamber were removed by cotton swab and the migrated cells on the bottom of the insert membrane were fixed using 100% methanol for 15 min at room temperature. Next, the cells were stained with 0.15% crystal violet in 10% ethanol for 15 min. After washing and drying, the insert membranes were photographed under a light microscope. For elution, the stained cells were resolved in 10% acetic acid for 5 min. ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used for measuring of O.D value (570 nm).
Human prion-Specific siRNA (nucleotide 502e522) [27] were purchased from Genolution Pharmaceuticals Inc, Seoul, Korea. MDA-MB231 cells were transfected with RNAIMAX reagent (Invitrgen) using 1 mM Prion-siRNA or control siRNA. Prion expression was determined using real-time PCR at 48 h posttransfection.
2.10. Statistical analyses All experiments were repeated at least 3 times, unless indicated otherwise in figure legends. The levels of significance for comparison between samples were determined by t-test, using GraphPad Prism Software, version 5 (GraphPad Software, San Diego, CA, USA). The data in the graphs are expressed as the mean ± S.D. p < 0.05 was considered statistically significant.
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3. Results 3.1. Overexpression of PrPc enhances migration and invasion of MCF-7 cells To investigate the function of PrPc in breast cancer, we made stable transfectant of MCF-7 cells overexpressing PrPc by antibiotics selection. Ectopic expressing of PrPc was confirmed at mRNA level by RT-PCR (Fig. 1A) and enhanced PrPc expression was confirmed by western blotting (Fig. 1B). To access the function of PrPc, we first measure the proliferation of PrPc overexpressing cells and found that PrPc expression did not influence the cell proliferation rates of MCF-7 cells (Fig. 1C). Next, we observed the invasion and migration of MCF-7 cells. Invasion was determined by number of cells infiltrated on the matrigel coated transwell membrane. As shown in Fig. 1D, number of invaded PrPc -overexpressing MCF-7 cells on bottom side of the filter is almost two fold of control cells. Would healing assay was performed to measure the migration of PrPc -overexpressing and control MCF-7 cells. After 16 h, PrPc -overexpressing cells move longer distance than control cells (Fig. 1E). Overall, these results show that overexpression of PrPc increase the invasion and migration of MCF-7 breast cancer cells. 3.2. Upregulation of MMP-9 by prion MMP-9 is a member of metalloprotease family, highly expressed in various cancers and regulates the invasion and migration of cancer cells by its gelatinase activity [25,26]. RT-PCR analysis showed that increase of mRNA of MMP-9 in prion-overexpressed cells (Fig. 1F) although there are no difference of expression level TIMP-1. Increase of MMP-9 expression was determined in western blotting and the gelatinase activity was also increased in PrPcoverexpressing MCF-7 cells (Fig. 1G). These data suggest that augmented expression of MMP-9 could be involved in increase of invasion and migration of MCF-7 cells by PrPc -overexpression. 3.3. NF-kB pathway is crucial for the PrPc-induced MMP-9 upregulation, invasion and migration in breast cancer cells To investigate the mechanism of MMP-9 increase in PrPc-overexpressed cells, we analyzed proximal promoter of MMP-9 gene by reporter assays. It has been reported that MMP-9 proximal promoter is regulated through two major regulatory transcription factors AP-1 and NF-kB binding sites [28,29]. To elucidate the transcription factor binding sites in MMP-9 promoter, we compared transcriptional activity of mutant promoters that include mutations at the each transcription factor binding sites and wildtype promoter (Fig. 2). Ectopic expression of PrPc induced prompter activity of wild type MMP-9 proximal promoter and mutated promoter with null AP-1 binding sites (Fig. 2A, C). But nonfunctional mutation in NF-kB promoter abolished the enhanced promoter activity by ectopic expression of PrPc (Fig. 2B). In addition, augmented NF-kB activity was confirmed by reporter assay using NF-kB reporter construct (Fig. 2D) ref. Increased expression of activated form of p65 protein of NF-kB transcription factor (Fig. 2E) and phosphorylated IkBa (Fig. 2G) by PrPc-expression showed that prion-expression activated the classical NF-kB activation pathway in MCF-7 breast cancer cells. Chromatin immunoprecipiation analysis with p65 protein of NF-kB transcription factor showed that increased association of NF-kB transcription factor with proximal promoter of MMP-9 by PrPc-overexpression (Fig. 2G). Overall, there results suggested that PrPc-overexpression enhanced MMP-9 proximal promoter activity through the activation of NF-kB classical pathway.
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3.4. Activation of ERK is involved in increased MMP-9 promoter activity and gelatinase activity To investigate the role of MAP kinase pathways on the regulation of MMP-9 expression by PrPc, we tested activation of MAP kinases by PrPc -overexpression by western blotting (Fig. 3A). Among MAP kinases, only phosphorylation of ERK was augmented by PrPc -expression. Inhibitor of ERK, PD98059 repressed the activation of MMP-9 promoter by PrPc -overexpression although other MAP kinase inhibitors did not influenced the MMP-9 activity (Fig. 3B). PD98059 also reduced enhanced gelatinase activity of MMP-9 (Fig. 3C). These results showed that ERK pathway is crucial for the regulation of MMP-9 activity with PrPc. 3.5. Silencing of PrPc inhibits migration and invasion of breast cell To investigate role of PrPc in breast cancer cells, we reduced the expression of PrPc by the knockdown with siRNA in MDA-MB231 cells which have high endogenous expression of PrPc [15]. SiRNA transfection successfully deleted the expression of PrPc (Fig. 4A). Knockdown of PrPc reduced the ERK activity (Fig. 4B), MMP-9 promoter activity (Fig. 4C), gelatinase activity (Fig. 4D), invasion (Fig. 4E), and migration (Fig. 4F) of MAD-MB-231 cells. These results were reversal of the PrPc -overexpression in MCF7 cells and further suggested the role of PrPc in invasion and migration of breast cancer cells through the NK-kB and ERK pathway. 4. Discussion PrPc has been extensively studied because its conformation isoform PrPSc for its critical role in the pathogenesis of transmissible spongiform encephalopathies. Physiological function of normal PrPc expression is involved in many important biological processes, such as cell adhesion, migration, proliferation, differentiation, neuroprotection, and inflammation. etc [4e6]. However, the precise physiological role of PrPc remains incompletely understood in various type of normal and tumor cells. Metastasis is the spread of a cancer from one organ or part to another part of the body. Approximately 10e15% of breast cancer patients develop distant metastases and its metastases is the main cause of death. Therefore, the development of therapeutic modality to regulate breast cancer metastasis are of great importance. PrPc have been implicated in breast cancer progression. Expression of PrPc are associated with a poor progression of breast cancer [15]. Several in vitro studies implicated that PrPc was involved in protection of breast cancer cells from anti-cancer drugs [8,9,16e18]. However, role of PrPc in metastasis of breast cancer has not been well elucidated. In the current study, we investigated the role of the PrPc in invasion of breast cancer cells and elucidated the mechanisms of PrPc -mediated induction of invasion. Silencing of PrPc repressed the Paclitaxel-induced invasion of multidrug-resistant breast cancer cells [17]. These data suggests a possible relationship between the invasiveness of breast cancer cells and the expression of PrPc. Furthermore, PrPc promotes invasion of gastric [30] and colorectal [31] cancer cells. Using overexpression and knock-down of PrPc, the present study reveals PrPc as an important target in the invasion and migration of breast cancer cells. MMP-9, also known as, Gelatinase B, plays a major role in tumor progression [32]. Pathologic investigations demonstrate that the expression of MMP-9 is strongly associated with breast cancer metastasis and is negatively correlated with patient survival [33e35]. In this study, we demonstrated that overexpression of PrPc increases MMP-9 expression in breast cancer cell. In breast cancer, higher expression of PrPc are associated with a poor prognosis [15].
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Fig. 2. NF-kB is required for the PrPc-induced MMP-9 upregulation, invasion and migration. AeD) Reporter gene assays with wild type and mutated MMP-9 proximal promoters. PrPc-overexpressing MCF-7 cells or control were transfected with (A) pGL-MMP-9 (wt), (B) pGL-MMP-9 (mut NF-kB), or (C) pGL-MMP-9 (mut AP-1U) reporter plasmids, or (D) with reporter plasmids containing tandem NF-kB binding. Cells were then harvested and assayed for luciferase activity. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01). E) Expression of p65 in nuclear fraction was measured by western blotting in control and Prionoverexpressing MCF-7 cells. F) Expression of IkBa and phosphorylated IkBa were measured by western blotting. G) Chromatin immunoprecipitation using the anti-NF-kB (p65) was performed on chromatin extracted from control or MCF-7-overexpressing cells, and the specific MMP-9 promoter regions were amplified by PCR.
Fig. 3. ERK activation is crucial for the PrPc-induced MMP-9 upregulation. A) Activation of p38, JNK and ERK were measured by western blotting in control and PrPc -overexpressing MCF-7 cells. B) PrPc -overexpressing cells transfected with pGL MMP-9 were treated with 20 mM of SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor) for 24 h. Cell were then harvested and assayed for luciferase activity. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01). C) Prion-overexpressing cells were treated with 20 mM of SP600125, SB203580, and PD98059 for 24 h. Conditioned media was collected, and the gelatinolytic activity of secreted MMP-9 was determined by gelatin zymogram assay. Fig. 1. Increasing of invasion and migration of MCF-7 cells by PrPc -overexpression. MCF-7 cells were transfected with control or PrPc expression vectors to establish stable transfectants. AeC) The expression of PrPc was determined by semi-quantitative RT-PCR (A), western blot analysis (B). C) Proliferation of transfectants was measured by cell counting measured with hemocytometer D) PrPc -overexpressing or control cells were subjected to the in vitro invasion assay. After 24 h, the cells on the bottom side of the filter were counted and imaged under a phase-contrast microscope. E) Migration of PrPc-overexpressing or control cells were subjected to the wound healing assay. A confluent monolayer of control or PrPc-overexpressing MCF-7 cells was wounded, and wound closure was photographed after 16 h and distance of migration was measured. F) Total RNAs were isolated from control- and PrPc-overexpressing cells and the expression of MMP-9 and TIMP-1 were measured by semi-quantitative RT-PCR. G) Conditioned media was collected and analyzed for the expression of and MMP-9 by western blot analysis. Zymography assays were performed, and the gelatinolytic activity of active MMP-9 (92 kDa) was visualized as clear bands against a blue background of stained gelatin. Bars show the mean ± S.D. of three independent experiments, performed in triplicate. *P < 0.01, significantly different from control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Inhibition of MMP-9 by silencing of PrPc with siRNA in MDA-MB231 cells. A) PrPc expression was determined by western blotting in MDA-MB-231cells transfected with human prion siRNA, and controls. B) Activation of ERK was determined by western blotting in siRNA-transfected MDA-MB-231cells and controls. C) Luciferase activity with pGL MMP-9 was assayed in siRNA-transfected MDA-MB-231cells and controls. D) Conditioned media was collected, and MMP-9 activity in MDA-MB-231 cells was determined by gelatin zymography and western blot analysis. E) Cells transfected with siRNA for Prion were subjected to in vitro invasion assays in MDA-MB-231cells. After 24 h, the cells on the bottom side of the filter were counted. F) A confluent monolayer of MDA-MB-231 cells transfected with siRNA and control was wounded, and wound closure was photographed after 24 h and distance of migration was measured. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01).
However, correlation between MMP-9 and PrPc expression in patient breast cancer sample and its prognostic value is remained to be investigated for future study. AP-1 and NF-kB binding sites in the proximal promoter of MMP9 mediate transcriptional regulation of MMP9 expression [28,29]. In the present study, mutational analyses of the promoter region revealed that PrPc regulates the MMP-9 promoter activity only through NF-kB binding site. Increase phosphorylation and degradation of IkB and nuclear localization and association of p65 to proximal promoter of MMP-9 suggest that NF-kB binding sites on MMP-9 promoter is regulated by classical NF-kB signal transduction pathway [36]. It has been demonstrated that extracellular regulated kinase (ERK) was an important downstream effector of PrPc in neuronal cells, non-neuronal cells [37] and gastric cancer cells [30]. Our results showed PrPc also activated ERK in breast cancer cell. ERKdependent activation of MMP-9 promoter by PrPc is a novel finding. This result suggest the possible application of ERK inhibitor as therapeutic use for breast cancers that has high expression level of PrPc. Taken together, our results provide the first evidence that PrPc promotes breast cancer cell invasion through the upregulation of MMP-9. We also provide evidence for the importance of the NF-kB and ERK pathway a in MMP-9 upregulation and invasion of breast cancer cells by PrPc. Our findings may provide a molecular basis for
the promoting role of PrPc in breast cancer progression and suggest PrPc as the possible novel therapeutic target in breast cancer. Conflicts of interest We have no conflicts of interest to declare. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) and funded by the Korean government (20080062286; 2015R1D1A1A02061508) and a grant (2011-0794) from the Asan Institute for Life Sciences (Seoul, Korea). References [1] A. Aguzzi, C. Sigurdson, M. Heikenwaelder, Molecular mechanisms of prion pathogenesis, Annu. Rev. Pathol. 3 (2008) 11e40. [2] C. Soto, Prion hypothesis: the end of the controversy? Trends Biochem. Sci. 36 (2011) 151e158. [3] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13363e13383. [4] A. Aguzzi, F. Baumann, J. Bremer, The prion's elusive reason for being,, Annu. Rev. Neurosci. 31 (2008) 439e477. [5] M. Mehrpour, P. Codogno, Prion protein: from physiology to cancer biology, Cancer Lett. 290 (2010) 1e23. [6] M.K. Bakkebo, S. Mouillet-Richard, A. Espenes, W. Goldmann, J. Tatzelt, M.A. Tranulis, The cellular prion protein: a player in immunological
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cellular prion protein regulates invasion and migration of breast cancer cells through MMP-9 activity Minchan Gil a, Yun Kye Kim d, Kyung-Eun Kim a, Wook Kim c, Chan-Sik Park b, **, Kyung Jin Lee d, * a
SIS Immunology Research Institute, Sookmyung Women's University, Seoul, South Korea Department of Pathology, Cell Dysfunction Research Center, University of Ulsan College of Medicine, Seoul, South Korea Department of Molecular Science and Technology, Ajou University, Suwon 443-749, South Korea d Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 138-736, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2015 Accepted 7 January 2016 Available online 9 January 2016
Function of cellular prion protein (PrPc) in cancer progression has not been elucidated yet. Ectopic expression of PrPc increases the invasion and migration of breast cancer cell line, MCF-7 cells. Overexpressed PrPc increases matrix metalloprotease-9 (MMP-9) expression by enhancing association of NF-kB in promoter of MMP-9 gene and ERK signaling in MCF-7 cells. Whereas, silencing of PrPc by siRNA suppresses ERK activation and MMP-9 expression resulting the down-regulation of MD-MB231 cell migration and invasion. Overall, these results suggest that PrPc contribute the breast cancer invasion and migration via MMP-9. © 2016 Elsevier Inc. All rights reserved.
Keywords: Prion Matrix metalloprotease-9 Migration Invasion Breast cancer
1. Introduction Cellular prion protein (PrPc) is a glycosylphosphatidylinositollinked extracellular membrane glycoprotein, which has been extensively studied for its critical role in the pathogenesis of transmissible spongiform encephalopathies. A pathogenic conformational isoform PrPSc is an infectious agent in Prion disease [1e3]. PrPc is highly expressed in neurons along with wild range of expression in various tissues. Physiological function of normal PrPc expression has been investigated in various cell types has been shown to be involved in many important biological processes, such as cell adhesion, migration, proliferation, neuroprotection, and inflammation [4e6]. However, the precise physiological role of PrPc remains incompletely understood in various types of cancer cell. PrPc is highly expressed in various cancer cells including glioma [7], breast cancer [8,9], prostate tumor [10], gastric cancer [11], colorectal cancer [12], pancreatic cancer [13], and osteosarcoma
Abbreviations: AP-1, activator protein-1; Ab, antibody; PrPc, cellular prion protein; MMP-9, matrix metalloprotease-9. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.-S. Park), [email protected] (K.J. Lee). http://dx.doi.org/10.1016/j.bbrc.2016.01.038 0006-291X/© 2016 Elsevier Inc. All rights reserved.
[14]. In breast cancer, higher expressions of PrPc are associated with a poor prognosis [15]. The role of PrPc to breast cancer cells has been investigated in several studies [8,9,16e18]. Overexpression of PrPc protects the breast cancer cell from TNF-a induced cell death by preventing Bax-mediated cytochrome C release [8,16]. Silencing of PrPc sensitizes Adriamycin-resistance carcinoma cells to TRAILmediated cell death [9]. Additionally, PrPc mediates the Paclitaxel-induced invasion of breast cancer cells [17]. By contrast, knockdown of PrPc in breast cancer cells are more resistance to doxorubicin treatment [18]. However, it remains unclear whether PrPc contributes to metastasis of breast cancer cells. Metastasis is composed of complex, multi-step process that involves the separation of tumor cells from original tumor site, intravasation, extravasation, and the settlement of tumor cells at a secondary site [19]. The invasiveness of tumor cells is critical in the all metastasis processes. Several proteases, including matrix metalloproteinase's (MMPs), cysteine proteinases, and serine proteases, are required for invasion because invasion requires altering extracellular matrix to permit the egress and ingress of cancer cells [20e22]. Among the MMPs, MMP-9 are thought to be directly involved in cancer cell growth and progression during metastasis [23,24]. Increased MMP-9 activity is observed in various tumor cells such as human breast cancer, lung cancer and ovarian cancer [25,26].
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In this study, we investigated the role of PrPc on invasion and migration of breast cancer cell lines by overexpression and knockdown of PrPc. Our results suggest that PrPc increase the migration and invasion in of breast cancer by activating NF-kB and ERK pathway. 2. Materials and methods 2.1. Establishment of prion-overexpressing stable transfectants PrPc overexpressing plasmids (kindly provided by Dr. Greene LE) were transfected into MCF-7 and MDA-MB231 (American Type Culture Collection, Manassas, VA) cells with lifopectamine 2000 (Invitrogen, Eugene, OR) and subjected to antibiotic selection for 1 month. The stable transfectant with highest PrPc expression were selected for further experiments. 2.2. RNA preparation, semi-quantitative RT-PCR Total RNA was extracted from the cells using NucleoSpin (Macherey-Nagel, Düren, Germany). The PCR amplification protocol was 30 cycles of 94 C for 30 s, 56 C for 30 s, and 72 C for 1 min. Amplified products were resolved by 1.5% agarose gel electrophoresis. The primers that were used are 50 -GGCAGTGACTATGAGGA eCCGTTAC-30 and 50 -GGCTTGACCAGCATCTCAGGTCTA-30 for human prion; 50 -TGACAGCGACA- AGAAGTG-30 and 50 -CAGTGAAGCGGTACATAGG-30 for MMP-9; 50 -CAATTCCGACCTCGTCATCA-30 and 50 -TCAGAGCCTTGGAGGAGCT-30 for TIMP-1 50 GATGATATCGC- CGCGCTCGTCGTCGAC-30 and 50 -GCCAGGTCCAGACGCAGGATGGCATG-30 and 50 AGCCAGGTCCA0 GACGCAGGATGGCATG-3 for b-actin. 2.3. Western blot analysis Cells were harvested and washed 3 times with cold phosphatebuffered saline (PBS). The cytoplasmic and nuclear protein fractions were extracted using NE-PER extraction reagents according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL). Nuclear protein extracts or whole protein extracts were used for Western blot analysis. Western blotting was performed using antiprion, anti-MMP-9, anti-JNK/ERK/p38, anti-phospho-JNK/ERK/p38, anti-Lamine and anti-actin antibodies. Protein samples were heated at 95 C for 5 min and were analyzed using SDSePAGE. Immunoblot signals were developed by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA).
2.5. Cell migration assay MCF-7 cells were seeded on collagen-covered 35-mm plates at a density of 0.4 106 per plate. After 24 h, the medium was replaced by 0.5% FBS containing DMEM, in each plate; three areas were scratched, creating three gaps of similar widths. Immediately thereafter, and at 24 h later, phase-contrast images of the plates were obtained with a CCD camera connected to an Olympus fluorescent microscope (X 10 objective). The region imaged at zero time was marked to enable us to photograph the same area at different times, thus allowing examination of a specific population of migrating cells. The widths of gaps were measured by Image-Pro Plus software. The data acquired from the three scratches on each plate were averaged to obtain the mean gap width.
2.6. Gelantin zymography MMP-9 enzymatic activity was accessed by gelatin zymography. The culture media were subjected to non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 0.2% gelatin-containing gels. After electrophoresis, gels were washed in 2.5% Triton X-100 to remove the SDS; incubated for 22 h at 37 C in 50 mm TriseHCl, pH 7.5, containing 0.15 M NaCl, 10 mm CaCl2, and 0.02% NaN3; and stained with 0.1% coomassie brilliant blue R250.
2.7. Transient transfection and luciferase assay The luciferase assays were performed using the luciferase assay system and the b-galactosidase assay system (Promega, Madison, WI). Transfection of MCF-7 cells was performed using Lipofectamine 2000 (Invitrogen). A standard transfection reaction used 1 mg of DNA, including the MMP-9 proximal promoter reporter constructs (kindly gifted from Dr. Wolfgang Eberhardt), internal control construct (pM1-b-gal), and expression construct (pCDNA3.1 or pCDNA3.1-prion). Luciferase activities were normalized with respect to b-galactosidase activities. The data shown are a representative of three independent experiments.
2.8. Chromatin immunoprecipitation Chromatin immunoprecipitation was carried out with Chromatin Immunoprecipitation Assay Kit (Upstate Biotechnology Inc., Lake Placid, NY) according to the manufacture's protocol.
2.9. RNA interference 2.4. Cell invasion assay Invasion assay was performed using a Transwell chamber (Costar, Cambridge, MA, USA) with 8 mm pore polycarbonate filters precoated with Matrigel (BD Biosciences, Franlin Lakes, NJ, USA). Briefly, 5 104 cells were harvested and suspended in 100 ml serum-free media and added to the upper chambers of the transwell system. Medium containing 10% FBS was placed into the lower chamber. After 24 h, non-migrated cells and media on the upper chamber were removed by cotton swab and the migrated cells on the bottom of the insert membrane were fixed using 100% methanol for 15 min at room temperature. Next, the cells were stained with 0.15% crystal violet in 10% ethanol for 15 min. After washing and drying, the insert membranes were photographed under a light microscope. For elution, the stained cells were resolved in 10% acetic acid for 5 min. ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used for measuring of O.D value (570 nm).
Human prion-Specific siRNA (nucleotide 502e522) [27] were purchased from Genolution Pharmaceuticals Inc, Seoul, Korea. MDA-MB231 cells were transfected with RNAIMAX reagent (Invitrgen) using 1 mM Prion-siRNA or control siRNA. Prion expression was determined using real-time PCR at 48 h posttransfection.
2.10. Statistical analyses All experiments were repeated at least 3 times, unless indicated otherwise in figure legends. The levels of significance for comparison between samples were determined by t-test, using GraphPad Prism Software, version 5 (GraphPad Software, San Diego, CA, USA). The data in the graphs are expressed as the mean ± S.D. p < 0.05 was considered statistically significant.
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3. Results 3.1. Overexpression of PrPc enhances migration and invasion of MCF-7 cells To investigate the function of PrPc in breast cancer, we made stable transfectant of MCF-7 cells overexpressing PrPc by antibiotics selection. Ectopic expressing of PrPc was confirmed at mRNA level by RT-PCR (Fig. 1A) and enhanced PrPc expression was confirmed by western blotting (Fig. 1B). To access the function of PrPc, we first measure the proliferation of PrPc overexpressing cells and found that PrPc expression did not influence the cell proliferation rates of MCF-7 cells (Fig. 1C). Next, we observed the invasion and migration of MCF-7 cells. Invasion was determined by number of cells infiltrated on the matrigel coated transwell membrane. As shown in Fig. 1D, number of invaded PrPc -overexpressing MCF-7 cells on bottom side of the filter is almost two fold of control cells. Would healing assay was performed to measure the migration of PrPc -overexpressing and control MCF-7 cells. After 16 h, PrPc -overexpressing cells move longer distance than control cells (Fig. 1E). Overall, these results show that overexpression of PrPc increase the invasion and migration of MCF-7 breast cancer cells. 3.2. Upregulation of MMP-9 by prion MMP-9 is a member of metalloprotease family, highly expressed in various cancers and regulates the invasion and migration of cancer cells by its gelatinase activity [25,26]. RT-PCR analysis showed that increase of mRNA of MMP-9 in prion-overexpressed cells (Fig. 1F) although there are no difference of expression level TIMP-1. Increase of MMP-9 expression was determined in western blotting and the gelatinase activity was also increased in PrPcoverexpressing MCF-7 cells (Fig. 1G). These data suggest that augmented expression of MMP-9 could be involved in increase of invasion and migration of MCF-7 cells by PrPc -overexpression. 3.3. NF-kB pathway is crucial for the PrPc-induced MMP-9 upregulation, invasion and migration in breast cancer cells To investigate the mechanism of MMP-9 increase in PrPc-overexpressed cells, we analyzed proximal promoter of MMP-9 gene by reporter assays. It has been reported that MMP-9 proximal promoter is regulated through two major regulatory transcription factors AP-1 and NF-kB binding sites [28,29]. To elucidate the transcription factor binding sites in MMP-9 promoter, we compared transcriptional activity of mutant promoters that include mutations at the each transcription factor binding sites and wildtype promoter (Fig. 2). Ectopic expression of PrPc induced prompter activity of wild type MMP-9 proximal promoter and mutated promoter with null AP-1 binding sites (Fig. 2A, C). But nonfunctional mutation in NF-kB promoter abolished the enhanced promoter activity by ectopic expression of PrPc (Fig. 2B). In addition, augmented NF-kB activity was confirmed by reporter assay using NF-kB reporter construct (Fig. 2D) ref. Increased expression of activated form of p65 protein of NF-kB transcription factor (Fig. 2E) and phosphorylated IkBa (Fig. 2G) by PrPc-expression showed that prion-expression activated the classical NF-kB activation pathway in MCF-7 breast cancer cells. Chromatin immunoprecipiation analysis with p65 protein of NF-kB transcription factor showed that increased association of NF-kB transcription factor with proximal promoter of MMP-9 by PrPc-overexpression (Fig. 2G). Overall, there results suggested that PrPc-overexpression enhanced MMP-9 proximal promoter activity through the activation of NF-kB classical pathway.
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3.4. Activation of ERK is involved in increased MMP-9 promoter activity and gelatinase activity To investigate the role of MAP kinase pathways on the regulation of MMP-9 expression by PrPc, we tested activation of MAP kinases by PrPc -overexpression by western blotting (Fig. 3A). Among MAP kinases, only phosphorylation of ERK was augmented by PrPc -expression. Inhibitor of ERK, PD98059 repressed the activation of MMP-9 promoter by PrPc -overexpression although other MAP kinase inhibitors did not influenced the MMP-9 activity (Fig. 3B). PD98059 also reduced enhanced gelatinase activity of MMP-9 (Fig. 3C). These results showed that ERK pathway is crucial for the regulation of MMP-9 activity with PrPc. 3.5. Silencing of PrPc inhibits migration and invasion of breast cell To investigate role of PrPc in breast cancer cells, we reduced the expression of PrPc by the knockdown with siRNA in MDA-MB231 cells which have high endogenous expression of PrPc [15]. SiRNA transfection successfully deleted the expression of PrPc (Fig. 4A). Knockdown of PrPc reduced the ERK activity (Fig. 4B), MMP-9 promoter activity (Fig. 4C), gelatinase activity (Fig. 4D), invasion (Fig. 4E), and migration (Fig. 4F) of MAD-MB-231 cells. These results were reversal of the PrPc -overexpression in MCF7 cells and further suggested the role of PrPc in invasion and migration of breast cancer cells through the NK-kB and ERK pathway. 4. Discussion PrPc has been extensively studied because its conformation isoform PrPSc for its critical role in the pathogenesis of transmissible spongiform encephalopathies. Physiological function of normal PrPc expression is involved in many important biological processes, such as cell adhesion, migration, proliferation, differentiation, neuroprotection, and inflammation. etc [4e6]. However, the precise physiological role of PrPc remains incompletely understood in various type of normal and tumor cells. Metastasis is the spread of a cancer from one organ or part to another part of the body. Approximately 10e15% of breast cancer patients develop distant metastases and its metastases is the main cause of death. Therefore, the development of therapeutic modality to regulate breast cancer metastasis are of great importance. PrPc have been implicated in breast cancer progression. Expression of PrPc are associated with a poor progression of breast cancer [15]. Several in vitro studies implicated that PrPc was involved in protection of breast cancer cells from anti-cancer drugs [8,9,16e18]. However, role of PrPc in metastasis of breast cancer has not been well elucidated. In the current study, we investigated the role of the PrPc in invasion of breast cancer cells and elucidated the mechanisms of PrPc -mediated induction of invasion. Silencing of PrPc repressed the Paclitaxel-induced invasion of multidrug-resistant breast cancer cells [17]. These data suggests a possible relationship between the invasiveness of breast cancer cells and the expression of PrPc. Furthermore, PrPc promotes invasion of gastric [30] and colorectal [31] cancer cells. Using overexpression and knock-down of PrPc, the present study reveals PrPc as an important target in the invasion and migration of breast cancer cells. MMP-9, also known as, Gelatinase B, plays a major role in tumor progression [32]. Pathologic investigations demonstrate that the expression of MMP-9 is strongly associated with breast cancer metastasis and is negatively correlated with patient survival [33e35]. In this study, we demonstrated that overexpression of PrPc increases MMP-9 expression in breast cancer cell. In breast cancer, higher expression of PrPc are associated with a poor prognosis [15].
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Fig. 2. NF-kB is required for the PrPc-induced MMP-9 upregulation, invasion and migration. AeD) Reporter gene assays with wild type and mutated MMP-9 proximal promoters. PrPc-overexpressing MCF-7 cells or control were transfected with (A) pGL-MMP-9 (wt), (B) pGL-MMP-9 (mut NF-kB), or (C) pGL-MMP-9 (mut AP-1U) reporter plasmids, or (D) with reporter plasmids containing tandem NF-kB binding. Cells were then harvested and assayed for luciferase activity. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01). E) Expression of p65 in nuclear fraction was measured by western blotting in control and Prionoverexpressing MCF-7 cells. F) Expression of IkBa and phosphorylated IkBa were measured by western blotting. G) Chromatin immunoprecipitation using the anti-NF-kB (p65) was performed on chromatin extracted from control or MCF-7-overexpressing cells, and the specific MMP-9 promoter regions were amplified by PCR.
Fig. 3. ERK activation is crucial for the PrPc-induced MMP-9 upregulation. A) Activation of p38, JNK and ERK were measured by western blotting in control and PrPc -overexpressing MCF-7 cells. B) PrPc -overexpressing cells transfected with pGL MMP-9 were treated with 20 mM of SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor) for 24 h. Cell were then harvested and assayed for luciferase activity. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01). C) Prion-overexpressing cells were treated with 20 mM of SP600125, SB203580, and PD98059 for 24 h. Conditioned media was collected, and the gelatinolytic activity of secreted MMP-9 was determined by gelatin zymogram assay. Fig. 1. Increasing of invasion and migration of MCF-7 cells by PrPc -overexpression. MCF-7 cells were transfected with control or PrPc expression vectors to establish stable transfectants. AeC) The expression of PrPc was determined by semi-quantitative RT-PCR (A), western blot analysis (B). C) Proliferation of transfectants was measured by cell counting measured with hemocytometer D) PrPc -overexpressing or control cells were subjected to the in vitro invasion assay. After 24 h, the cells on the bottom side of the filter were counted and imaged under a phase-contrast microscope. E) Migration of PrPc-overexpressing or control cells were subjected to the wound healing assay. A confluent monolayer of control or PrPc-overexpressing MCF-7 cells was wounded, and wound closure was photographed after 16 h and distance of migration was measured. F) Total RNAs were isolated from control- and PrPc-overexpressing cells and the expression of MMP-9 and TIMP-1 were measured by semi-quantitative RT-PCR. G) Conditioned media was collected and analyzed for the expression of and MMP-9 by western blot analysis. Zymography assays were performed, and the gelatinolytic activity of active MMP-9 (92 kDa) was visualized as clear bands against a blue background of stained gelatin. Bars show the mean ± S.D. of three independent experiments, performed in triplicate. *P < 0.01, significantly different from control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Inhibition of MMP-9 by silencing of PrPc with siRNA in MDA-MB231 cells. A) PrPc expression was determined by western blotting in MDA-MB-231cells transfected with human prion siRNA, and controls. B) Activation of ERK was determined by western blotting in siRNA-transfected MDA-MB-231cells and controls. C) Luciferase activity with pGL MMP-9 was assayed in siRNA-transfected MDA-MB-231cells and controls. D) Conditioned media was collected, and MMP-9 activity in MDA-MB-231 cells was determined by gelatin zymography and western blot analysis. E) Cells transfected with siRNA for Prion were subjected to in vitro invasion assays in MDA-MB-231cells. After 24 h, the cells on the bottom side of the filter were counted. F) A confluent monolayer of MDA-MB-231 cells transfected with siRNA and control was wounded, and wound closure was photographed after 24 h and distance of migration was measured. Each bar represents the mean ± S.D. calculated from three independent experiments. *Significantly different from control (P < 0.01).
However, correlation between MMP-9 and PrPc expression in patient breast cancer sample and its prognostic value is remained to be investigated for future study. AP-1 and NF-kB binding sites in the proximal promoter of MMP9 mediate transcriptional regulation of MMP9 expression [28,29]. In the present study, mutational analyses of the promoter region revealed that PrPc regulates the MMP-9 promoter activity only through NF-kB binding site. Increase phosphorylation and degradation of IkB and nuclear localization and association of p65 to proximal promoter of MMP-9 suggest that NF-kB binding sites on MMP-9 promoter is regulated by classical NF-kB signal transduction pathway [36]. It has been demonstrated that extracellular regulated kinase (ERK) was an important downstream effector of PrPc in neuronal cells, non-neuronal cells [37] and gastric cancer cells [30]. Our results showed PrPc also activated ERK in breast cancer cell. ERKdependent activation of MMP-9 promoter by PrPc is a novel finding. This result suggest the possible application of ERK inhibitor as therapeutic use for breast cancers that has high expression level of PrPc. Taken together, our results provide the first evidence that PrPc promotes breast cancer cell invasion through the upregulation of MMP-9. We also provide evidence for the importance of the NF-kB and ERK pathway a in MMP-9 upregulation and invasion of breast cancer cells by PrPc. Our findings may provide a molecular basis for
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