Enhanced expression of polysialic acid correlates with malignant phenotype in breast cancer cell lines and clinical tissue samples
- Authors:
- Published online on: October 27, 2015 https://doi.org/10.3892/ijmm.2015.2395
- Pages: 197-206
Abstract
Introduction
Sialic acids, which are mainly attached to the terminal of N-glycans, are abundantly present in a number of organisms, particularly during embryonic development. Sialic acids have been found to mediate various cellular processes in mammals. Polysialic acid (PSA) is a negatively charged homopolymer composed of α-(2,8)-linked sialic acid residues, which is involved in brain development and certain psychiatric disorders, such as schizophrenia (1–3). PSA is barely expressed during postnatal development, but may be 're-expressed' in a number of types of tumor (such as lung cancer, pancreatic cancer, neuroblastoma and glioma), where it modulates cell adhesion, migration and invasion (4–7). The biosynthesis of PSA is catalyzed by two Golgi-resident polysialyltransferases, ST8SiaIV (PST) and ST8SiaII (STX) (8). It has been demonstrated that the mRNA levels of PST and STX are closely associated with the development of pancreatic cancer (9) and non-small cell lung cancer (10). In patients with breast cancer (BC), serum levels of PSA and sialyltransferases have been shown to be positively associated with the presence of malignant tumors and negatively with responses to anticancer treatment (11). However, to the best of our knowledge, few studies to date have examined the expression of PSA and polysialyltransferases in tissues from patients with BC.
Epithelial-mesenchymal transition (EMT) plays a role in tissue repair and pathological processes, notably in tissue fibrosis and facilitates tumor metastasis (12). During EMT, epithelial cancer cells acquire a mesenchymal phenotype and express mesenchymal markers, such as vimentin (13,14). EMT is therefore a potential target for the development of novel immunotherapeutic approaches. Sialic acids, the ligands for the sialic-acid-binding immunoglobulin-like lectins (Siglec) family of cell adhesion molecules, appear to be involved in regulating the immune response (15). Exploiting the enzymatic permissiveness of sialic acids has been successfully used for the immunotargeting of cancer cells (16). Thus, PSA may be a useful immunotherapeutic target for cancer cells undergoing EMT. The cell lines, NMuMG and MCF10A, derived respectively from mouse mammary glands and non-malignant human breast epithelial tissues, are commonly used for studies on EMT induced by treatment with transforming growth factor-β1 (TGF-β1) (17). The elucidation of changes in PSA and polysialyltransferase levels during EMT in these two cell lines will help to guide future studies on BC.
The major goals of the present study were to i) use a fluorescence-labeling method followed by HPLC to quantify the PSA expression levels in normal and malignant breast epithelial cells, in cells undergoing TGF-β1-induced EMT, and in 24 clinical BC specimens; ii) evaluate the effects of PSA on cell motility and migration; iii) examine the correlation between PSA expression with that of the related polysialyltransferase, PST, in clinical BC samples.
Materials and methods
Antibodies and reagents
The following antibodies were used: mouse anti-E-cadherin IgG2a (1:50,000; #610181); mouse anti-β-catenin IgG1 (1:10,000; #610153) (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin IgG1 (1:1,000; sc-59987) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); mouse anti-vimentin IgG1 (1:1,000; V5255); mouse anti-PSA-neural cell adhesion molecule (NCAM) IgM 5A5 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); anti-β-tubulin I IgG1 (1:1,000,000; T7816) (Sigma, St. Louis, MO, USA); and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5,000; A0216) (Beyotime Institute of Biotechnology, Haimen, China).
The reagents used in this study were as follows: TGF-β1 (BD Biosciences), PNGase F (New England BioLabs, Inc., Ipswich, MA, USA), urea, DL-dithiothreitol, iodoacetamide, tetrachloroaurate (AuCl4), Sepharose 4B, methanol, tunicamycin and 1-butanol (Sigma).
Cell lines and cell culture
Mouse mammary epithelial cells (NMuMG), mouse mammary carcinoma cells (4T1), normal human breast cells (MCF10A) and human mammary carcinoma cells (MDA-MB-231; MB-231) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco, Carlsbad, CA, USA) in a humidified 5% CO2 atmosphere at 37°C. The NMuMG cells were cultured in DMEM containing 10 µg/ml insulin (Sigma). The MCF10A and MB-231 cells were cultured in DMEM containing 1% sodium pyruvate (Solarbio, Beijing, China). The 4T1 cells were cultured in RPMI-1640.
Ethics approval
All procedures performed in experiments involving human participants were carried out in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Patients and tissue samples
BC tissues were obtained from 20 patients with TNM stage I, II and III BC, and 4 normal breast tissue samples were obtained from surgical or autopsy specimens performed at the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China). All the tissues were snap-frozen in liquid nitrogen and stored at −70°C until use. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. The present study was approved by the Research Ethics Committee of Jiangnan University (Wuxi, China).
Western blot analysis
The cells were cultured in 6-well plates, washed with phosphate-buffered saline (PBS), harvested, homogenized in T-PER lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 10 U/ml aprotinin, and centrifuged at 14,000 × g at 4°C for 15 min. The super-natants were mixed with loading buffer (which contained 1% β-mercaptoethanol) and heated at 100°C for 10 min. Proteins were loaded on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk, incubated with primary antibody overnight at 4°C, probed with appropriate HRP-conjugated secondary antibody, visualized using the Pro-Light HRP kit (Tiangen Biotech Co., Ltd., Beijing, China), and photographed using a Molecular Imager Chemi DOC™XRS+ system (Bio-Rad, Richmond, CA, USA).
Separation of glycans
Total proteins from the tissues were isolated using an E.Z.N.A. DNA/RNA/Protein Isolation kit (Omega Bio-Tek, Doraville, CA, USA) according to the manufacturer's instructions. Glycans on proteins were released by PNGase F and desalted as previously described (18,19). The glycan sample was lyophilized and dissolved in methanol/H2O (1:1, v/v) for further analysis.
Fluorescence labeling of free sialic acids for 1,2-diamino-4,5-methylenedioxybenzene (DMB) and HPLC analysis
Free sialic acids were labeled with DMB using a Sialic Acid Fluorescence Labeling kit (Takara Bio Inc., Otsu, Japan), according to the instructions provided by the manufacturer. Briefly, the cell lysate was hydrolyzed with 2 M acetic acid and labeled with DMB, as described in a previous study (20). A 10-µl aliquot of each sample was loaded on a Zorbax SB-C18 column (4.6×250 mm) (Agilent Technologies, Inc., Santa Clara, CA, USA) and eluted with methanol/acetonitrile/water (8:7:85, v/v/v) on a Waters e2695 HPLC system with a fluorescence detector (excitation, 373 nm; emission, 448 nm). Free sialic acids were quantified based on peak areas obtained from a defined standard.
Reverse transcription-quantitative (real-time) polymerase chain reaction (RT-qPCR)
Total RNA from the cells was isolated using an RNApure Tissue kit (CWBIO, Beijing, China) and RNA from human tissues was isolated using an E.Z.N.A. DNA/RNA/Protein Isolation kit (Omega Bio-Tek) according to the manufacturer's instructions. First-strand cDNA was synthesized using a ReverTra Ace-α® kit (Toyobo, Osaka, Japan). Primers were designed using DNAman software as follows: mouse STX (167 bp) sense, 5′-CTTGGATGCGGAGAAGGAT and anti-sense, 5′-GGCACAAGTCTGGAAATGCT; mouse PST (126 bp) sense, 5′-GCGAACTGCCTATCCATCAC and antisense, 5′-TCACAGAATCTGGTGGCAAG; human STX (141 bp) sense, 5′-TCAGAACCAGAACCCAGTCA and antisense, 5′-CGACAGTCAGTTTCAAAGCC; human PST (106 bp) sense, 5′-ACTGA AAGTGCGAACTGCCT and antisense, 5′-GAGAAGACCTGTGCTGGGTC; mouse γ-tubulin (107 bp) sense, 5′-ATCTA CCTGTCGGAGCATGG and antisense, 5′-GCCTCCCGA TCTATGATGTC; and human β-actin (232 bp) sense, 5′-CTTCC TGGGCATGGAGTC and antisense, 5′-GCCGATCCACA CGGAGTA. Semi-quantitative PCR was performed as follows: 94°C, 4 min; 94°C, 45 sec, 60°C, 45 sec, 72°C, 15 sec for 30 cycles; 72°C, 5 min; quantitative PCR (qPCR) was performed using Ultra SYBR Mixture (Cat. no. CW0957; CWBIO) on a CFX96 Real-Time PCR detection system (Bio-Rad). The transcriptional levels of target genes were quantified using the 2−ΔΔCt method, as previously described (21) and expressed as the means ± SD from triplicate experiments.
Cell motility assay
Phagokinetic gold sol assay was performed as previously described (22,23). Briefly, 1.8 ml of a 14.5 mM AuCl4 solution and 6 ml of a 36.5 mM Na2CO3 solution were added to 11 ml distilled H2O, heated to a boil, and then 1.8 ml of 0.1% formaldehyde was added. Colloidal gold solution (gold sol) was added to 24-well plates, and the plates were blocked with filter-sterilized 1% BSA, as previously described (22). Cells (2×103) in complete culture medium were seeded onto the gold sol-coated wells and incubated for 18 h. Images were captured using an inverted microscope (model NA0.3OWD72; Sunny Optical Technology, Ningbo, China). The track areas of 50 cells were measured using the ToupView imaging system, as previously described (24) and expressed as square pixels.
Gene silencing with small interfering RNA (siRNA)
Duplexes of 21 nucleotides of human and mouse STX siRNA target sequences (hSTXi or mSTXi) and negative control siRNA (NC), without homology to other known human and mouse genes, were designed and synthesized by Invitrogen (Carlsbad, CA, USA) as follows: hSTXi, 5′-GCCUGGAGAUAUUAUUCA UTT (sense); and mSTXi, 5′-CCUGAAGCCUGGAGACAU UTT (sense). siRNA (30 pmol) was transfected using Lipofectamine 2000 reagent (3 µl) (Invitrogen) and the 4T1 or MB-231 cells were examined 24 h following transfection. The suppression of the expression of STX was verified by semi-quantitative and quantitative RT-PCR.
Transwell migration assay
The cells were cultured in 6-well plates and treated with or without TGF-β1 (5 ng/ml) for 48 h. Tunicamycin (4 µg/ml) was added together with TGF-β1 into 6-well plates, and the NMuMG or MCF10A cells were cultured for an additional 48 h, as previously described (25,26). The cells (5×104) were plated in an upper Transwell insert (12/24-well Transwell; 8 µm polycarbonate membrane; Costar, Corning, NY, USA) in DMEM or RPMI-1640 medium containing 0.1 ml of 0.2% BSA (RuiTaibio, Beijing, China). A total of 0.6 ml of DMEM or RPMI-1640 medium supplemented with 10% FBS, serving as a chemoattractant, was deposited in the lower chamber. Following incubation for 16 h at 37°C in a 5% CO2 atmosphere, the cells were washed with PBS and fixed with cold 4% buffered paraformaldehyde. The cells in the upper Transwell filter were removed with a cotton wool tip and stained with crystal violet. The Transwells were rinsed with deionized water and air-dried. The filters were photographed, and the cells in 5 random optical fields were counted to determine migration, as previously described (27,28).
Data analysis
Data were statistically analyzed using the Prism 5 software programs as previously described (29). Differences between the means were assessed by paired or unpaired Student's t-test, and P-values <0.05 were considered to indicate statistically significant differences.
Results
PSA level is higher in malignant cancer cells than in normal cells
As EMT is one of the main mechanisms involved in the development of BC metastasis (30), the expression of EMT-related markers in malignant 4T1 and MB-231 cells, in comparison with non-malignant NMuMG and MCF10A cells was determined by western blot analysis (Fig. 1A). The decreased expression of E-cadherin and β-catenin, as well as the increased expression of vimentin, were observed in the 4T1 and MB-231 cells. Tumor progression towards metastasis is often depicted as a multistage process in which malignant cells spread from their original site to colonize distant organs through acquired mobility and migration ability (31,32). In this experiment, increased motility (Fig. 1B) and migration (Fig. 1C) were observed in malignant 4T1 and MB-231 cells compared with normal NMuMG and MCF 10A cells.
In view of the findings that PSA is re-expressed in many types of tumor, we thus compared PSAs on N-glycans in these 4 cell lines. PSAs display unique modifications of N-glycans, particularly of NCAM (33). As shown in Fig. 2A, PSAs on N-glycans were hydrolyzed under mild acidic conditions, and free sialic acids (reflecting PSA levels) were detected using a fluorescence-labeling method followed by HPLC. In comparison with the NMuMG or MCF10A cells, the PSA level was slightly higher in the 4T1 cells and significantly higher in the MB-231 cells (Fig. 2B). These findings suggest that a high PSA expression is associated with the enhanced invasiveness and metastasis of BC cells.
PSA expression level is increased in NMuMG and MCF10A cells undergoing EMT
The EMT process has been shown to be associated with cancer cell motility and metastasis. The non-malignant NMuMG and MCF10A cells were treated with TGF-β1 to induce EMT, as previously described (34). The treated cells displayed a spindle-like, elongated morphology (Fig. 3A), a reduced β-catenin expression, an increased vimentin expression, and exhibited the typical 'cadherin switch' from E-cadherin to N-cadherin (Fig. 3B). The TGF-β1-treated NMuMG cells displayed an enhanced motility and migration, whereas the TGF-β1-treated MCF10A cells exhibited only enhanced migration (Fig. 3C and D). The levels of free sialic acid released from N-glycan as a function of dose and time were examined in the NMuMG cells following treatment with TGF-β1. The NMuMG cells treated with 2 and 5 ng/ml TGF-β1 exhibited free sialic acid levels approximately 2.5- and 4-fold higher, respectively, than those of the untreated cells (Fig. 4A). In order to examine the effects of high levels of PSA on cell behavior, 5 ng/ml TGF-β1 was used in the following experiment. The PSA levels were higher in the TGF-β1-treated NMuMG cells at various time points (Fig. 4B). Similar results were obtained for the TGF-β1-treated MCF10A cells (Fig. 4C). Moreover, a high expression of polysialylated NCAM (PSA-NCAM) was observed in the NMuMG and MCF10A cells undergoing EMT (Fig. 4D).
PSA facilitates cell migration in malignant breast cells and in normal breast cells undergoing EMT
In view of the correlation of high PSA levels with the facilitation of cell migration and cell motility during EMT, we examined the possibility that the downregulation of PSA reverses EMT in malignant cells. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of PST and STX, two major polysialyltransferases responsible for PSA synthesis. The 4T1 and MB-231 cells exhibited high levels of STX, but undetectable levels of PST (data not shown). The silencing of PSA by the suppression of STX resulting in the decreased expression of PSA-NCAM (Fig. 5A and B), or by treatment with tunicamycin (which blocks N-glycan synthesis), significantly decreased the migration of both malignant cell lines (4T1 and MB-231) (Fig. 5C). The downregulation of PSA resulted in the decreased motility of the malignant BC cells (MB-231), but not of the 4T1 cells (Fig. 5C).
To further investigate the role of PSA during EMT, we used tunicamycin to inhibit N-glycan synthesis in normal cells undergoing EMT. The typical 'cadherin switch' from E-cadherin to N-cadherin and the enhancement of cell migration in the TGF-β1-treated MCF10A and NMuMG cells were reversed by the presence of tunicamycin (Fig. 6).
PSA expression level is related to the BC stage
The patient characteristics and PSA levels in the normal tissues (n=4) and malignant tissues (n=20) are listed in Tables I and II. The PSA levels were lower (pmol level) in the normal tissues and higher in the malignant tumor tissues (Fig. 7A). The difference in the PSA levels between the normal tissues and advanced-stage BC tissues was significant (P<0.0001). The PSA level was consistently higher in the tumor tissues compared to the normal controls. In comparison with the controls, the PSA level was highest in the samples obtained from patients with TNM stage III BC (n=6, P<0.0001), followed by stage II (n=8, P<0.0001) and stage I (n=6, P=0.0085), suggesting that an increased PSA expression correlates with disease progression (Fig. 7B).
![]() | Table IIAssociation of disease characteristics with polysialyltransferase (STX or PST) gene expression in malignant BC patients. |
PST mRNA level may serve as an indicator for human BC
The aberrant expression of polysialyltransferases is often observed in malignant tumors and has thus been considered as a novel target for detection or treatment of metastatic cancer (35). As an enhanced level of PSA was observed in our clinical BC samples, we wished to examine the expression of polysialyltransferases between normal tissues and malignant tissues. We used semi-quantitative and quantitative RT-PCR to examine STX and PST expression at the mRNA level in BC tissues (Table I). STX was widely and highly expressed in all tissues examined (n=24) with no significant differences when compared with the normal controls (Fig. 7C and D). However, PST expression was significantly increased in the malignant tumor tissues compared to the normal tissues (Fig. 7C and E). PST was expressed distinctively in the tumor samples obtained from patients with stage I (n=6, 50%), II (n=8, 75%), and III (n=6, 83%) BC (Table II and Fig. 7E). The PST enzyme is clearly expressed in high-stage BC tissues, but not in normal tissues. The difference in the PST level between the normal tissues (n=4) and high-stage/lymph node-positive tissues (n=6, stage III) from patients with BC was significant at r2=0.8, indicating that PST expression is increased in BC tissues and correlates with cancer progression.
Discussion
Highly sialylated glycans on the surface of cancer cells often correlate with tumor invasiveness and metastasis (33). α2,6-sialylation on the cell surface has been shown to affect the adhesion of MDA-MB-435 breast carcinoma cells (36). Another study revealed that negatively charged PSA led to the increased motility of pancreatic carcinoma cells and subsequently reduced cell adhesion (9). In this study, to assess PSA levels on N-glycans, we applied a DMB-labeling method followed by HPLC, to quantify PSA expression at the pmol level. Our results revealed an increased motility of breast carcinoma 4T1 and MB-231 cells in comparison with the non-malignant NMuMG and MCF10A cells. PSA levels on N-glycans were higher in malignant mouse and human cell lines.
Altered PSA levels were observed in the NMuMG and MCF10A cells undergoing TGF-β1-induced EMT. The EMT process in our models involved increased motility and migration. PSA expression in the cells undergoing EMT increased in a TGF-β1 dose- and time-dependent manner. We further investigated the role of PSA in malignant and normal breast cells undergoing EMT by downregulating PSA biosynthesis. The partial removal of PSA from malignant BC cells using siRNA decreased migration and motility. The treatment of normal breast cells with tunicamycin, an inhibitor of N-glycan synthesis, indicated a similar role for PSA during EMT. The suppression of PSA did not cause significant alterations in the typical EMT markers (aside from reduced cell migration) in these cell models. Our findings suggest that PSA has a greater effect on cell migration than cell motility in malignant cells.
A previous study demonstrated an increased expression of PSA and sialyltransferases in the sera of patients with BC (11). In this study, we directly examined the expression of PSA and polysialyltransferase genes in human BC tissues, and evaluated the correlation between the PSA expression level and disease stage in 24 tissue samples from patients with BC and normal controls. PSA expression was higher in the clinical BC specimens than in the normal tissues obtained from patients with BC. Taken together, our findings indicate that PSA expression in BC tissues is related to the TNM stage.
The polysialyltransferases STX and PST differentially and dependently contribute to the re-expression of PSA based on the histological origin of the tumor (37) and are regarded as anti-metastatic therapeutic targets. It has been demonstrated that STX is more important than PST, due to its dominant expression in cells and tissues (35). In the present study, we observed a marked increase in PST mRNA expression. The PST gene exhibited a higher expression in the advanced-stage cases than in normal tissues, whereas the STX gene was widely and highly expressed in all clinical cases. Polysialyltransferase expression appears to be the basis for the aberrant expression of sialylated structures on N-glycans from BC tissue samples. The PST gene plays a crucial role in BC progression and is a potential target for molecular therapy of BC.
In conclusion, our data demonstrate that PSA is highly expressed in malignant BC cells and in normal mouse mammary and human breast cell lines undergoing EMT. The effects of PSA on cell migration were more pronounced than those on cell motility in malignant BC cells. PSA expression correlates with the TNM stage in human BC samples and depends on PST activity, whereas STX is expressed consistently in both normal and BC tissues. The detailed functions of these two polysialyltransferases remain unclear, however. A previous in vitro study found that PST forms more highly polysialylated N-glycans than does STX (38). Our finding of a higher PST expression in advanced-stage BC may reflect the presence of more highly polysialylated N-glycans in these patients. It remains to be clarified whether the increased PSA expression in BC results from synthesis by PST alone or by PST in cooperation with STX.
Acknowledgments
This study was supported by the National Science Foundation for Young Scientists of China (no. 81201572), the Natural Science Foundation of Jiangsu Province, China (no. BK2012113), the Fundamental Research Funds for the Central Universities (no. JUSRP51319B), the Jiangsu Province 'Six Summit Talent' Foundation (no. 2013-SWYY-019), and the 111 Project (no. 111-2-06). The authors are grateful to Dr S. Anderson for English editing of the manuscript.
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