Introduction

Breast cancer has become a primary cause of cancer mortality among women worldwide, and breast cancer metastasis accounts for the majority of cancer-related recurrences and deaths. During metastasis, cancer cells need to complete multistep cell-biological processes, which include matrix proteolysis and invasion into the tumor-associated stroma. Then, tumor cells enter into the lumina of lymphatic or blood vessels and survive in circulation. The final step involves in cancer cells crossing from the lumina vessel into the tissue parenchyma, forming micrometastases in distant organs, and finally proliferating within the microenvironment, leading to the formation of macroscopic metastases [1, 2]. Single-cell RNA sequencing has shown that breast cancer-derived liver and brain metastases exhibit a more complicated intratumoral heterogeneity and immunosuppressive ecosystem, which contribute to immune evasion and tumor malignancy [4, 5]. Snail, in particular, is a well-studied transcription factor and a key regulator of EMT and cancer metastasis involved in multiple mechanisms [6]. Previous studies indicate that Snail promotes tumor progression by conferring tumor cells with stemness traits and promoting tumor cells’ chemoresistance, recurrences, metastasis and cancer-associated fibroblast activation [7,8,9,39]. In this study, we report that the expression of CHST2 contributes to breast cancer metastasis. We discovered breast cancer cell migration and metastasis reduced after depleted CHST2, while cell proliferation is not affected. Conversely, overexpression of CHST2 increased breast cancer cell migration and metastasis in vitro and in vivo. Moreover, cell migration assays indicated that CHST2 is required in Snail-mediated cell metastasis. It is known that in many cellular types overexpression of Snail induces EMT and cell migration, but how much role of EMT contributes to cell migration is unknown. Besides, other mechanisms exist for Snail-mediated cell migration, including formation of lamellipodia upon activation Rac1 and the promotion of neurite outgrowth in prostate cancer[13, 40]. In this study, we discovered CHST2 acts as a novel downstream target to promote Snail-mediated cell migration by increasing cell surface MECA79 antigen synthesis. In addition, sodium chlorate treatment or catalytically inactive mutant can efficiently abolish CHST2 or Snail-mediated cancer cell migration. Those results suggest that CHST2 acts as a critical role in breast cancer metastasis and its function in mediating sialyl LewisX sulfation is responsible for breast cancer metastasis.

The biosynthesis of 6-sulfo sialyl LewisX is a multienzyme catalytic process of glycosylation and sulfation, the mechanisms of glycosylation and post-translational modifications of carbohydrate structures are extremely intricate. Sulfation at the non-reducing C-6 of GlcNAc is essential for L-selectin binding and MECA79 epitope synthesis [41]. Here, we focused on the ubiquitously expressed enzyme CHST2 which catalyzed the transfer of sulfate groups to GlcNAc structures. Our experiments clearly show that CHST2 is responsible for MECA79 antigen synthesis in breast cancer cells. Furthermore, Snail-CHST2 axis mediated sulfation of sialyl LewisX is probably the downstream target to facilitate breast cancer metastasis. The results account for clinical studies which showed ectopic expression of MECA79 antigen on tumor specimens are linked with poor prognosis and metastasis [36, 42, 43]. The carbohydrate antigen sialyl LewisX is a terminal carbohydrate structure which is associated with tumor malignancy. Sialyl LewisX can serve as a tumor marker and alter cancer cell phenotypes by increasing cancer cell motility and proliferation through E-selectin–mediated cancer cell adhesion to vascular endothelial cells or E-selectin-independent pattern [44, 45]. 6-sulfo sialyl LewisX is the sulfation form of sialyl LewisX, in which the GlcNAc is sulfated by specific sulfotransferases. The sulfation modification alters the binding and expression properties of sLeX, making it a ligand for L-selectin, a cell surface receptor expressed on leukocytes [24, 46]. Overall, both sialyl LewisX and 6-sulfo sialyl LewisX are carbohydrate antigens that are associated with tumor malignancy and play important roles in cancer progression and metastasis [47]. However, 6-sulfo sialyl is less well understood in terms of its role in cancer biology. In our study, we tried to elucidate the biological role of 6-sulfation sialyl LewisX in breast cancer and discovered cell surface 6-sulfo sialyl LewisX synthesis mediated by CHST2 is associated with breast cancer metastasis and may be a potential novel breast cancer metastatic marker.

Current findings demonstrate Snail induces CHST2 transcription, which results in cell surface 6-sulfo sialyl LewisX synthesis increase and facilitates migration and metastasis of breast cancer cells. Nevertheless, further studies must be done in the future to elucidate the detailed mechanisms of ectopic MECA79 antigen expression in breast cancer. Previous studies have reported that glycoproteins carrying 6-sulfo sLeX include GlyCAM-1, CD34, MAdCAM-1, PODXL and EMCN, a class of proteins that have mucin-like domains, which act as scaffoldings for O-linked oligosaccharides [48, 49]. Therefore, further investigations will be carried out to identify the glycoproteins and to elaborate on the mechanisms of MECA79 synthesis in breast cancer.

Indeed, studies have demonstrated that 6-sulfo sLeX synthesis in L-selectin ligands requires two 6-sulfotransferases, N-acetylglucosamine-6-O-sulfotransferase-2 (GlcNAc6ST-2, CHST4) which specifically restricted expressed in HEV and ubiquitously expressed CHST2 [22]. In the present study, we have revealed that the expression of CHST2 is induced by Snail in breast cancer cells, while CHST4 expression is not affected by Snail. It has been reported that CHST2 and CHST4 mediated sulfation of sLeX are tissue- and substrate-specific. Specifically, Kenji Uchimura et al. reported CHST2 is the major sulfotransferase enzyme in Peyer’s patches, while in peripheral and mesenteric lymph nodes, CHST4 plays a key role in the synthesis of 6-sulfo sLeX. Those results suggest that CHST2 and CHST4 play complementary roles in 6-sulfo sLeX synthesis in lymph nodes [38]. Here, we reported that Snail-induced expression of CHST2 in breast cancer cells is responsible for 6-sulfo sLeX synthesis, which mediates breast cancer metastasis.

In conclusion, our observations shed light on the molecular mechanisms underlying Snail transactivating CHST2 transcription and expression and elaborate the role of Snail-CHST2 axis in mediating sulfation of sialyl LewisX on the cell surface during breast cancer metastasis. These results provide new insights into Snail function in cancer metastasis. Furthermore, this novel sulfation pathway regulated by Snail may be a promising therapeutic strategy by specifically targeting the MECA79 antigen. In addition, the ectopic expression of the MECA79 antigen will contribute to the diagnosis and treatment of breast cancer.

Materials and methods

Cells and cell culture

The human breast cancer cell lines MDA-MB-231, MCF-7 and non-tumorigenic mammary cells MCF-10A were originally obtained from the American Type Culture Collection (ATCC). Human embryonic kidney cell line 293T, MDA-MB-231 and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine and penicillin (50 U/ml)/streptomycin (50 μg/ml). MCF-10A cells were maintained in DMEM/F12 supplemented with 5% horse serum, insulin (10 μg/ml), hydrocortisone (0.5 μg/ml), EGF (20 ng/ml), cholera toxin (100 ng/ml) and penicillin/streptomycin. The cell lines were authenticated by DNA fingerprinting in the Shanghai Jiao Tong University Analysis Core and were cultured in a 37 °C water-saturated 5% CO2 humidified chamber.

Plasmids

The human CHST2 cDNA was subcloned into pCDH-CMV-MCS-EF1-Puro-vector by PCR technique with EcoR I and Not I restriction enzyme sites. The pCDH-Flag-CHST2-N475A variant were cloned from pCDH-Flag-CHST2 by point mutation of the amino acid residue 475 N into A. The CHST2 gene promoter spanning from −638 to +426 was generated from genomic DNA of the human embryonic kidney cell line 293T by PCR and subcloned into pGL3.0-Luc basic vector, generating the pGL3-CHST2-Luc reporter constructs. The pLU-Flag-Snail plasmid has been previously described [34]. The short hairpin RNA sequences of pLKO.1- shCHST2 were obtained from GE-Life science and are listed in Table 1.

Table 1 Primer list.
Full size table

RNA extraction and RT-PCR analysis

Total RNA was extracted following standard protocols with TRIzol reagent (Ambion, Carlsbad, CA, USA). Complementary DNAs were synthesized with 3 μg of total RNA using iScript cDNA Synthesis Kit (Fermentas, San Jose, CA, USA). The detailed procedures of RNA extraction and qRT-PCR were previously described [50]. The qRT-PCR assays were performed with SYBR green reagent (ABI 7500 Fast).

Western blots and antibodies

Protein samples were prepared from various cells at 60~80% confluency. The cells were washed in ice-cold PBS, and then lysed in ice-cold lysis buffer containing protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and cocktail, 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 1 mM EDTA. Cell lysates were then clarified by centrifugation, protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, USA). The antibodies were used in western blots as follows: CHST2 antibody (26027-1-AP, Proteintech, Chicago, IL, USA), Snail antibody (3879S, Cell Signaling Technology, Massachusetts, USA), anti-Flag (F3165, F7425, Sigma, St. Louis, MO, USA), Alpha Tubulin antibody (66031-1-Ig, Proteintech, Chicago, IL, USA), GAPDH antibody (60004-1-Ig, Proteintech, Chicago, IL, USA), Normal rabbit IgG (sc-2027, Santa Cruz).

Transfection, luciferase reporter assays and ChIP

For transfection, 293T cells were seeded at a density of 5 × 104 cells per well in 24-well plates. The β-galactosidase plasmids (10 ng) and pGL3-CHST2-Luc reporter (100 ng), along with Snail-encoding plasmids, were transiently transfected into the cells with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA). The following procedures were performed as described [34]. ChIP assay was performed according to published protocols [34] in MDA-MB-231 cells and MCF-10A stably expressing Snail cells with slight modifications. To prepare cells for ChIP, MDA-MB-231 cells and MCF-10A-Snail cells were grown in 100-mm plates to 70–80% confluence and fixed by the addition of 287 μl of 37% formaldehyde directly into 10 ml of growth medium to a final concentration of 1% for 15 min at room temperature. The cross-linking reaction was stopped by the addition of 625 μl of 2 M glycine in phosphate-buffered saline buffer at room temperature for 5 min. 1 × 107 cells were harvested, the chromatin was sonicated into fragments ranging from 500 to 800 bps in size, the sonication fragments were resolved on 2% agarose gels and visualized with ethidium bromide. The immunoprecipitated DNAs were amplified by real-time PCR with primer sets listed in Table 1.

Cell migration and invasion assays

Migration of breast cancer cells was determined using 24-well Boyden chambers (Corning) with 8 μm-inserts. The detailed procedures of migration assay were previously described [17, 51]. Exponentially growing MDA-MB-231, MCF-10A or MCF-7 cells were digested by trypsin and collected by centrifugation. The cells were resuspended in serum-free DMEM or DMEM/F12 blank medium and counted. 2.5 × 104 of MDA-MB-231 or 5 × 104 of MCF-10A or 4 × 104 of MCF-7 cells in 100 μl serum-free DMEM or DMEM/F12 blank medium were seeded on 8 μm-inserts, then 600 μl DMEM or DMEM/F12 complete growth medium were added to the bottom chamber as attractants. After incubation for 20–24 h, the cells were fixed with 4% paraformaldehyde and stained with crystal violet, non-migrated cells on the top of the chamber were removed gently with cotton swabs, then migrated cells were counted as per field of view under phase-contrast microscopy. For invasion assay, the matrigel matrix (Corning, #354234) was diluted to 250 μg/ml on ice, and 100 μl diluted matrigel was added into 8 μm-inserts. Then the transwell filter was placed in the 37 °C incubator for 2 h until the matrigel solidified. The following procedures are similar to migration assays. The number of migrated cells was calculated by the relative cell migration area through Image-J software.

Wound-healing assays

MCF-10A cells were stably expressed with Flag-CHST2 and Flag-CHST2-N475A individually, then, subconfluent growing cells were digested by trypsin and counted using hemocytometer, 5 × 105 cells were seeded onto a 6-well tissue culture plate. After incubating the seeded cells for 6 h at incubator with complete DMEM/F12 medium, the confluent monolayer cells were scratched in a straight line to create a “scratch” with a 10 μl pipette tip, removing the cell debris and smooth the edge of the scratch by washing the cells twice with 2 ml of the PBS, and then added 2 ml DMEM/F12 medium supplemented with 1% horse serum, insulin (2 μg/ml), hydrocortisone (0.1 μg/ml), EGF (4 ng/ml) and cholera toxin (20 ng/ml). And afterwards, creating markings on the outer bottom of the dish with an ultrafine tip marker. Then 0 h images were captured using a phase-contrast microscope. Place the plates in incubator at 37 °C under 5% CO2 for 24 h, the second images were captured of the same wounds at 24 h. The scratch areas were measured and presented as the percentage of scratch areas at 0 h (mean ± SD). The percent of migration was calculated as the migration value of the scratch areas at 24 h divided by the scratch areas at 0 h. Each sample was performed at least three times [52].

Cell proliferation and cell viability

Cell proliferation rate and cell viability were measured by cell counting kit-8 (C0038, Beyotime). For cell proliferation analysis, seed cells in a 96-well plate at a density of 1 × 103 cells per well in 100 μl of culture medium and incubate the plate in the incubator for ~12 h, then add 10 μl of CCK8 solution to each well and incubate another 1 hour, measure the absorbance at 450 nm every other day to determine the cell proliferation rate. For cell viability assay, 5 × 103 cells were seeded onto a 96-well plate, then incubate the plate for 4~6 h in a humidified incubator at 37 °C, 5% CO2, and after cells were treated with 0, 5 and 10 mM sodium chlorate for 24 h, add 10 μl of CCK8 solution to each well with another 1-hour incubation in the incubator, the absorbance of 450 nm was tested to determine cell viabilities.

Mouse experiments

Female BALB/c nude mice were purchased from SLAC laboratory Co. Ltd (Shanghai, China) and divided into two groups randomly. To establish in vivo lung metastatic mouse models of MDA-MB-231 cells, Exponentially growing MDA-MB-231 cells (labeled with luciferase reporter) were digested and resuspended in PBS buffer, then 1 × 106 of MDA-MB-231-sh-CHST2/sh-Vector or 5 × 105 of MDA-MB-231-Flag-CHST2/Vector cells in 100 μl of PBS suspensions were injected into the tail vein of 6-week-old mice. Lung micro-metastatic foci were analyzed by using the Xenogen IVIS Imaging System (PerkinElmer). Animal studies were conducted following the Institutional Animal Care and Use Committee of Shanghai in accordance with the National Research Council Guide for Care and Use of Laboratory Animals (SCXK, Shanghai 2007-0005). All mice were fed under specific pathogen-free (SPF) conditions. To ameliorate any suffering, mice were euthanized by CO2 inhalation.

Hematoxylin-eosin staining and immunohistochemistry assays

After lung metastatic mice models were analyzed at the endpoint, the mice were euthanized and lungs with metastatic foci were dissected and fixed with 5 ml of 4% paraformaldehyde. Tissues were then embedded in paraffin blocks, and sections were cut (4 μm) for H&E staining and immunohistochemistry. For immunohistochemical staining, lung sections were baked at 65 °C overnight, then de-paraffinized by three 10-min extractions in xylene, followed by a series of steps de-paraffinized using 100%, 100%, 90%, 90% and 75% ethanol for 5 min each. Then tissue slides are soaked in gently running tap water for 30 min. Afterwards, sections were transferred to boiling sodium citrate buffer (pH 6.0) for antigen retrieval for 30 min and then cooled to room temperature. Then, sections were pre-treated with 3% hydrogen peroxide for 15 min before blocking. Blocking was performed with 5% bovine serum albumin in PBS for 30 min at room temperature followed by primary antibody incubation overnight at 4 °C. Immunoreaction was detected using SABC-AP assay (Boster, SA1050) according to the manufacturer’s instructions. The sections were visualized under a microscope. The following primary antibodies were used: rat monoclonal MECA‐79 antibody (sc‐19602, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA).

Flow‑cytometric analysis

MDA-MB-231, MCF-7 and MCF-10A cells were harvested at a subconfluent stage, then the cells were collected by centrifugation for 5 min at 800 × g, at 4 °C, washed twice and resuspended in PBS containing 1% FBS at 1 × 106 cells/ml density. Subsequently, the cell suspensions were incubated with each mAb (MECA79 (rat mAb, Santa Cruz Biotechnology) or normal rat IgM (sc-3885, Santa Cruz Biotechnology) for 30 min on ice, followed by staining with APC-conjugated goat anti-rat IgM (A10540, Invitrogen, Carlsbad, CA, USA) or FITC-conjugated sheep anti-rat (PA1-28638, Invitrogen, Carlsbad, CA, USA) IgG for 30 min on ice, and then analyzed on a FACScan (CytoFLEX LX, Beckman Coulter, USA). Experiments were performed in triplicate.

Statistical analysis

Statistical analysis was performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and Graph Pad Prism 7.0 (Graph Pad Software, Inc., La Jolla, CA, USA) software. The independent student’s t tests were used for comparison between two groups. Data are presented as the mean ± SD. The correlation between the expression of Snail and CHST2 in breast tumor samples was evaluated by the Pearson rank correlation coefficient test. P < 0.05 was considered to indicate a statistically significant difference.