Demethylation of the Cosmc Promoter Alleviates the Progression of Breast Cancer Through Downregulation of the Tn and Sialyl-Tn Antigens.

BACKGROUND: Aberrant gene methylation in breast cancer is associated with an unfavorable prognosis. Besides, abnormal Cosmc can induce the expression of Tn and STn antigens. The present study aimed to investigate the roles of Cosmc promoter methylation in breast cancer through the regulation of Tn and STn antigens.
METHODS: The expression patterns of Cosmc and the Tn and STn antigens in breast cancer cell lines were determined. Cosmc was overexpressed to explore the effects of Cosmc on cell behavior, including the growth, migration, invasion, and apoptosis of breast cancer cells and tumor growth with in vitro and in vivo experiments. Afterwards, a methyltransferase and a methyltransferase inhibitor were used to alter the methylation status of Cosmc to explore the mechanisms related to Cosmc promoter methylation.
RESULTS: Cosmc was poorly expressed in breast cancer cells. Cosmc overexpression inhibited cell growth, migration, and invasion while promoting apoptosis in breast cancer cells in vitro and restraining tumor growth in vivo. Cosmc promoter methylation was found to decrease the levels of Cosmc and increased the expression of the Tn and STn antigens in breast cancer.
CONCLUSION: In conclusion, the demethylation of Cosmc mitigates breast cancer progression through the repression of the Tn and STn antigens, which provides evidence for therapeutic considerations for a novel target against breast cancer.

Introduction

Breast cancer is a frequently diagnosed female malignancy. It is also one of the three most common malignant tumors all over the world together with colon and lung cancer.1 Breast cancer has caught global attention since the number of women diagnosed with this disease has reached around 1.7 million in 2012.2 When compared with other cancers, breast cancer is more likely to occur in a younger population, with a median age of 61 years.3 Currently, the strategies for breast cancer treatment rely on tumor characteristics, tumor size, and location as well as tumor stage at the initial diagnosis.4 Soon, targeted therapies for breast cancer will present more opportunities for the improvement of current treatments that present complications such as overtreatment risk, excessive costs of cancer care, and diminishing returns.5 Moreover, risk-prediction models based on DNA methylation bring new hope for risk-targeted prevention and diagnosis of cancer diagnosis.6 For example, serum DNA methylation has been identified as a powerful biomarker for monitoring the prognosis and response of metastatic breast cancer.7 An enhanced understanding of the molecular mechanisms underlying tumorigenesis and progression of breast cancer is needed to provide new target candidates for novel therapeutic interventions.

Recently, Cosmc wild-type cells have been found to express the cell surface associated (MUC1), MUC1-Tn, and Sialyl-Tn (STn) that are correlated with breast cancer.8 Cosmc is a particular molecular chaperone in the endoplasmic reticulum, which is necessary for the folding of the enzyme and consequent movement of its single client T-synthase into the Golgi apparatus.9 Hypermethylation of the Cosmc promoter induces its epigenetic silencing, which repels Cosmc transcripts out of Tn4 cells that are short of the activity of T-synthase and express the Tn antigen.10 The Tn and STn antigens are predominant members of O-glycans, whose synthesis is associated with various glycosyltransferases, such as the T-synthase.11 The Tn antigen, an intermediate product of glycosylation, is a kind of carbohydrate antigen associated with tumor development, which derives from Cosmc and is absent in normal cells.12 The STn antigen is a short O-glycan with a sialic acid residue, whose synthesis is mainly controlled by the enzyme ST6GalNAc1 that accelerates the delivery of a sialic acid onto the Tn antigen.13 The expression of the STn antigen is correlated with an abnormally activated glycosylation pathway in tumor cells, which induces poor outcome and prognosis in cancer patients.14 However, the mechanisms of Cosmc in breast cancer remain largely unknown. Therefore, we aimed to elucidate the regulatory mechanisms of Cosmc in breast cancer progression related to the regulation of the Tn and STn antigens, to present a theoretical foundation for an enhanced understanding of breast cancer treatment.

Materials and Methods

Ethics Statement

The animal experiments were performed following the recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Chao-Yang Hospital.

Cell Culture and Treatment

The breast cancer cell lines MCF-7, MDA-MB-468, MDA-MB-453, MDA-MB-231, and normal breast cells were all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Next, the cell lines were cultured in 89% Dulbecco’s modified eagle’s medium (DMEM; Gibco Company, Grand Island, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco Company, Grand Island, NY, USA) and 10% fetal bovine serum (FBS) (Gibco Company, Grand Island, NY, USA). Western blot analysis was performed to measure the expression of Cosmc in the four breast cancer cell lines and normal breast cells. Cells in the logarithmic growth phase were detached with 1 mL of 0.25% trypsin (Shanghai Sangon Biotechnology Co. Ltd., Shanghai, China) for 3 min and neutralized with serum medium (Gibco Company, Grand Island, NY, USA). Afterwards, the cells (1 × 105 cells/mL) were inoculated into a 6-well plate for 24-h routine culture. When the cell confluence reached around 75%, the Cosmc vector (oe-Cosmc) (50 ng/mL, Shanghai GenePharma Co., Ltd., Shanghai, China) or its negative control (oe-NC) was introduced into the cells following the instructions of the Lipofectamine 2000 kit (Invitrogen Inc., Carlsbad, CA, USA). To alter the methylation status, the cells were treated with dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc., Waltham, MA, USA), methyltransferase M.SssI (Invitrogen, Carlsbad, California, USA), or 5µM 5-aza-2ʹ-deoxycytidine (5-aza-dc) (Beijing Biolab Technology Co., Ltd., Beijing, China).15

Western Blot Analysis

After trypsinization, the cells were lysed using enhanced radio-immunoprecipitation assay (RIPA) lysis buffer (Boster Biological Technology Co. Ltd., Wuhan, Hubei, China) supplemented with protease inhibitor. Next, the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster Biological Technology Co. Ltd., Wuhan, Hubei, China). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane that was later blocked with 5% bovine serum albumin (BSA; Shanghai Sangon Biotechnology Co. Ltd., Shanghai, China) at room temperature for 2 h to prevent non-specific binding. Then, the membrane was probed with the diluted mouse anti-human Cosmc primary antibody (sc-271829, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), the rabbit anti-human primary antibodies to Tn (ab233231, 1:1000, Abcam Inc., Cambridge, MA, USA), proliferating cell nuclear antigen (PCNA) (ab18197, 1:2000, Abcam), Ki67 (ab16667, 1:2000, Abcam), Bcl2 Associated X protein (Bax) (ab32503, 1:5000, Abcam), Bcl2 associated agonist of cell death (Bad) (ab90435, 1:5000, Abcam) and mouse anti-human antibody to STn (ab115957, 1:500, Abcam) overnight at 4°C. After 3 washes with phosphate-buffered saline with Tween-20 (PBST; Shanghai Sangon Biotechnology Co. Ltd., Shanghai, China), the membrane was probed with HRP-labeled secondary antibody, goat anti-rabbit ( ab6721, 1:2000, Abcam) or rabbit anti-mouse (ab6728, 1:2000) at room temperature for 1 h. After another 3 PBST washes, an enhanced chemiluminescence (ECL) solution (Merck Millipore, Billerica, MA, USA) was used for further color-developing. The Image J software was used for gray value analysis of each protein band with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal reference.

5-Ethynyl-2´-Deoxyuridine (EdU)

Cell viability was assessed using an EdU assay.16 The cells were inoculated into a 24-well plate, with 3 duplicate wells set for each group. Next, the cells were incubated with EdU (C10341-1, Guangzhou RiboBio Co., Ltd., Guangdong, China) for 2 h with a final concentration of 10 µmol/L. After the culture medium was removed, the cells were fixed with a PBS solution containing 4% paraformaldehyde at room temperature for 15 min and then washed with PBS containing 3% BSA twice. Next, the cells were permeabilized by adding 0.5% Triton-100 in PBS at room temperature for 20 min and washing again with PBS containing 3% BSA twice. The cells in each well were incubated with 100 µL of staining solution at room temperature for 30 min avoiding exposure to light and washed with PBS containing 3% BSA twice. The nuclei of the cells were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; C10341-1, Guangzhou Ribobio Biotechnology Co., Ltd., Guangzhou, China) for 5 min, followed by PBS washes and sealing. Lastly, 6–10 visual fields were randomly selected to observe and record the number of positive cells in each visual field under a fluorescence microscope (FM-600, Shanghai Pudan Optical Instrument Co., Ltd., Shanghai, China). The EdU labeling rate (%) = the number of positive cells/(the number of positive cells + the number of negative cells) × 100%.

Scratch Test

Next, a ruler and a marking pen were used to draw horizontal lines on the bottom of a 6-well plate at the interval of 0.5–1 cm, with at least 5 lines crossed per well. Then, the cells were inoculated into the plate at a density of 5 × 105 cells per well and cultured with a medium containing 10% FBS overnight. Subsequently, a 10 µL sterile pipette was used to scratch lines perpendicular to the horizontal lines on the bottom of the plate. Lastly, the scratch distance was measured and recorded under an optical microscope after 0 and 24 h of cell culture and the images were obtained under an inverted microscope to analyze cell migration.

Transwell Assay

Cell invasion was evaluated as previously outlined.17 The apical Transwell chamber was coated with Matrigel (Becton, Dickinson and Company, NJ, USA) and reacted at 37°C for 30 min to aggregate Matrigel into a gel. Next, the basolateral membrane was hydrated before the experiment. The cells were cultured in serum-free medium for 12 h, harvested and re-suspended by serum-free medium (1 × 105 cells/mL). Then, the basolateral chamber was added with medium containing 10% FBS and the Transwell chambers were added with 100 µL of cell suspension followed by incubation at 37°C for 24 h. The cells that did not invade the surface of the Matrigel membrane were gently removed with a cotton swab, and the rest of the cells were fixed in 100% methanol and stained with 1% toluidine blue (Sigma, St. Louis, MO, USA). Five fields were randomly selected to count the number of stained invasive cells under an inverted optical microscope (CarlZeiss Meditec AG, Oberkochen, Germany).

Flow Cytometry

Breast cancer cells were inoculated into a 6-well plate with medium without penicillin/streptomycin at a concentration of 1 × 106 cells/well. After 48 h of transfection, the harvested cells were washed twice and stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA). Next, cell apoptosis was detected using a flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) following the instructions of the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA).12

DNA Extraction and DNA Methylation in vitro

The genomic DNA from cultured cells was extracted and purified following the instructions from the PureLinkTM Genomic DNA Mini kit (Invitrogen Inc., Carlsbad, CA, USA). The DNA substrate was treated with the methyltransferase M.SssI including an optimized 10 × reaction buffer and 50 × accessory factors in an S-adenosylmethionine (SAM) solution. The methylation system was 40 μL, including 1 μg of DNA, 0.1 mM SAM, 1 × reaction buffer and 1 U of methyltransferase M.SssI. This mixed system was incubated at 37°C for 15 min and later treated for 20 min with a heat-treatment at 60°C before hydrolysis to deactivate the enzyme.

Methylation-Specific Polymerase Chain Reaction (MSP)

A DNA Methylation-GoldTM kit (D5005, Zymo Research, Irvine, CA, USA) was used to detect methylation of the Cosmc promoter region. The primer sequences used for MSP amplification were as follows: Cosmc-MD (5ʹ-GAGAGTTCGGGTTACGTAG-3ʹ) and Cosmc-MR (5ʹ-GTTAACGCACCACTCCGTTAC-3ʹ). The sequences of unmethylated primers were as follows: Cosmc-UD (5ʹ-GTGTTTGGGTTATGTAGGTTT-3ʹ) and Cosmc-UR (5ʹ-AACACACCACTCCATTACACTCC-3ʹ). The purified DNA was converted using a CT conversion reagent (D5005, Zymo Research, Irvine, CA, USA). Following degeneration and disulfate transformation, the DNA was desulfurated and purified. The PCR was performed with the reaction conditions as follows: a cycle of pre-denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 45 s, treatment at 56°C (methylated)/50°C (non-methylated) for 45 s, annealing at 72°C for 45 s and extension at 72°C for 45 s, and a final extension at 72°C for 10 min. Lastly, the PCR products were subjected to agarose gel electrophoresis, gel electrophoresis imaging and image analysis using an image analysis system (Invitrogen, Carlsbad, California, USA).

Bisulfite Sequencing PCR (BSP)

The bisulfite sequencing primers were designed, and the primer sequences were as follows: specific primer (F: 5ʹ-TTTTAAGAGAGGGAGGGGAGTTAGG-3ʹ; R: 5ʹ-TCCAAACAATAAAACTTCAAATCTCATTC-3ʹ). The steps and conditions of the PCR reaction were the same as above. The PCR products were selected for agarose gel electrophoresis. The target bands were cut under ultraviolet light and put into a 1.5 mL Eppendorf (EP) tube, and the recovery process was conducted according to the instructions of the agarose gel DNA recovery kit (Tiangen Bio-tech, Beijing, China). Then, the cloning reaction system was established, which consisted of 5.5 μL of PCR products (bisulfite sequencing prime amplification), 1 μL of T-easy, 1 μL of Ligase, and 7.5 μL of 2 × buffer (pGEM-T Easy Vector Systems, Promega, Madison, WI, USA), followed by an overnight reaction at 4°C. Subsequently, 10 μL of the ligation products were added into 100 μL of DH5a competent cells in a tube and evenly mixed. The products were placed on ice for 30 min, treated with thermal excitation at 42°C for 45 s, and incubated on ice for 1 min. Then, the products were added with 800 μL of Super Optimal broth with Catabolite repression (SOC) fluid medium, followed by oscillating-culture at 37°C for 60 min. The samples were then added with Luria-Bertani (LB) solid medium containing X-gal, isopropyl-β-D-thiogalactopyranoside (IPTG) and Amp, followed by oscillating-culture and amplification at 37°C. With at least 10 single bacterial colonies (white clones) selected, the PCR reactions for the clones were conducted using the BcaBEST sequencing Primers and MS universal primer, and the positive recombinants were screened using agarose gel electrophoresis, cultured overnight and treated with bacterial fluid sequencing.

Chromatin Immunoprecipitation (ChIP)

Following the instructions of a commercially available ChIP kit (Millipore, Billerica, MA, USA), the cells were fixed with 1% formaldehyde at room temperature for 10 min for stably cross-linking between intracellular DNA and proteins when the cell confluence reached 70% - 80%. Afterwards, the cells were ultrasonicated and the harvested fragments were centrifuged at 13,000 rpm and 4°C. The supernatant was collected into 3 tubes and then incubated with the antibody to RNA polymerase II in positive control, antibody to normal IgG in NC and mouse antibodies to Dnmt1 (ab13537, 1:100, Abcam), Dnmt3a (ab2850, 1:100, Abcam) and Dnmt3b (ab2851, 1:100, Abcam), respectively, overnight at 4°C. The endogenous DNA-protein compounds were precipitated with Protein Agarose/Sepharose. The supernatant was discarded after immediate centrifugation and the non-specific compounds were eluted. After de-crosslinking overnight at 65°C, the DNA fragments were purified by phenol/chloroform extraction and the expression of the Cosmc promoter was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Xenograft Tumor in Nude Mice

A total of 24 healthy nude mice aged 5–6 weeks (Beijing Institute of Pharmacology of Chinese Academy of Medical Science, Beijing, China) were acclimatized in separate cages of a specific pathogen-free (SPF) grade animal laboratory for a week, with a humidity of 60% - 65% at a temperature of 22°C - 25°C. The mice were provided with free access to food and water under a cycle of 12 h of light and darkness with their health conditions checked prior to the experiment. Meanwhile, the cell lines stably transfected with oe-Cosmc or oe-NC were constructed and made into a cell suspension which was subcutaneously inoculated into mice (12 mice per treatment). The tumor size was recorded during the growth process. After 5 weeks, the mice were euthanized to measure tumor weight and the expression of Tn, ST, Ki67, PCNA, Bax and Bad in tumor tissues.

Statistical Analysis

All data were analyzed using the Statistic Package for the Social Science (SPSS 19.0 statistical software (IBM Corp. Armonk, NY, USA). All experiments were repeated independently 3 times. The measurement data were described as mean ± standard deviation. The data distribution was tested for normality using the Kolmogorov–Smirnov method. Comparisons between two groups were performed using a t-test. If departure from normality and homogeneity of variance was not observed, a non-paired t-test was used. Comparisons among multiple groups for data with normal distribution were analyzed using the one-way analysis of variance (ANOVA) and a post-hoc test was conducted using Tukey’s test. The proliferation ability at different time points was analyzed by the repeated measures ANOVA. A value of p < 0.05 was considered statistically significant.

Results

Cosmc Is Poorly Expressed in Breast Cancer

The expression of the Tn/STn antigen was correlated with the clinicopathological features of tumors. Moreover, Cosmc gene mutation led to Tn/STn antigen exposure.18 Therefore, to explore the effects of Cosmc on breast cancer, Western blot analysis was conducted to measure the Cosmc protein level in breast cancer cell lines (MCF-7, MDA-MB-468, MDA-MB-453, and MDA-MB-231). The results showed that in contrast to normal breast epithelial cells, significantly decreased Cosmc protein levels were detected in the four breast cancer cell lines, having the lowest expression in MDA-MB-231 and the highest expression in MCF-7 (p < 0.05) (Figure 1). Hence, MDA-MB-231 and MCF-7 were selected for the subsequent experiments.

Figure 1

Cosmc is expressed poorly in breast cancer. Western blots and protein levels of Cosmc in normal breast epithelial cell line and breast cancer cell lines (MCF-7, MDA-MB-468, MDA-MB-453, and MDA-MB-231) were determined. *p<0.05 vs normal breast epithelial cell line. #p<0.05 vs MCF-7, MDA-MB-468 and MDA-MB-453. The above were measurement data and were expressed as mean ± standard deviation. Comparisons between two groups were performed using an independent sample t-test. All experiments were repeated independently 3 times.

Cosmc Overexpression Inhibits Growth, Migration, and Invasion While Promoting Apoptosis of Breast Cancer Cells

Cosmc gene mutation depletes T-synthase activity due to the loss of folding stability, thus causing carbohydrate chain synthesis obstruction and exposure to the Tn and STn antigens.18 To evaluate the effects of Cosmc on breast cancer, the MDA-MB-231 and MCF-7 cells were transfected with oe-Cosmc and the expression of Cosmc, T-synthase, and the Tn and STn antigens was measured by Western blot analysis. The results revealed that the transduction of oe-Cosmc significantly increased the expression of Cosmc and T-synthase while it greatly decreased the expression of the Tn and STn antigens in MDA-MB-231 and MCF-7 cells (p < 0.05; Figure 2A). Next, to further explore the effects of Cosmc on growth, migration, invasion, and apoptosis of breast cancer cells, EdU, scratch test, transwell assay, and flow cytometry were carried out. The results revealed that the transduction of oe-Cosmc led to significant reductions in cell growth, migration and invasion while causing a significant increase in apoptosis of MDA-MB-231 and MCF-7 cells (p < 0.05; Figure 2B, D, E, F). Meanwhile, Western blot analysis was performed to measure the expression of the proliferating-associated proteins (Ki67 and PCNA) as well as apoptosis-related proteins (Bcl-2, Bax and Bad). The results displayed that the expression of Bcl-2, Ki67, and PCNA was markedly reduced and that of Bax and Bad was significantly elevated following the transduction of oe-Cosmc in MDA-MB-231 and MCF-7 cells (Figure 2C, G). Together, these results supported the conclusion that Cosmc overexpression contributed to the inhibition of breast cancer cell growth, migration, and invasion and the promotion of apoptosis.

Figure 2

Elevation of Cosmc plays an inhibitory role in the growth, migration, and invasion and a promotive role in the apoptosis of breast cancer cells. MDA-MB-231 and MCF-7 cells were introduced with oe-Cosmc to generate breast cancer cell line overexpressing Cosmc. oe-NC served as a negative control. (A) protein bands and expression of Cosmc, T-synthase, Tn and STn antigens in MDA-MB-231 and MCF-7 detected by Western blot analysis. (B) proliferation (× 200) of MDA-MB-231 and MCF-7 cells measured by EdU assay. (C) protein bands and levels of proliferating-associated proteins (Ki67 and PCNA) in MDA-MB-231 and MCF-7 cells evaluated by Western blot analysis. (D) migration of MDA-MB-231 and MCF-7 cells measured by scratch test. (E) invasion (× 200) of MDA-MB-231 and MCF-7 cells measured by transwell assay. (F) apoptosis of MDA-MB-231 and MCF-7 cells measured by flow cytometry. (G) protein bands and levels of apoptosis-related proteins (Bcl-2, Bax and Bad) in MDA-MB-231 and MCF-7 cells evaluated by Western blot analysis. *p<0.05 vs the cells introduced with oe-NC. The above were measurement data and were expressed as mean ± standard deviation. Comparisons between two groups were performed using an independent sample t-test. All experiments were repeated 3 times.

Abbreviations: STn, Sialyl-Tn; EdU, 5-Ethynyl-2´-deoxyuridine; PCNA, proliferating cell nuclear antigen; Bcl-2, B-cell lymphoma-2; Bax, Bcl2 associated X; Bad, Bcl2 associated agonist of cell death; NC, negative control.

Demethylation of Cosmc Promoter Reduces Expression Tn and STn Antigens

As reported in a recent study, hypermethylation in the Cosmc promoter region has been shown to cause expression of the Tn and STn antigens.15 The MethPrimer website () was used to predict CpG islands in the Cosmc promoter region (Figure 3A) and design primers for MSP. Methylation was detected in specific sites of the Cosmc promoter in MDA-MB-231 and MCF-7 cells in comparison with normal breast epithelial cells. The results of BPS revealed that the number of Cosmc methylated sites was significantly increased in MDA-MB-231 and MCF-7 cells compared to normal breast epithelial cells, and a higher number of Cosmc methylated sites was found in MDA-MB-231 cells than in MCF-7 cells. Meanwhile, the DNA methylation status was determined in MDA-MB-231 and MCF-7 cells that were previously treated with methyltransferase M.SssI and methyltransferase inhibitor 5-aza-dc. The results displayed that compared with the treatment with DMSO, methylation in specific sites and elevation of the methylated sites were detected after treatment with M.SssI, and the opposite results were observed after the treatment with 5-aza-dc (p < 0.05; Figure 3B and C). Next, ChIP was performed to measure the enrichment of methyltransferase Dnmt1, Dnmt3a, and Dnmt3b in the Cosmc promoter region. The results showed that the MDA-MB-231 and MCF-7 cells exhibited significantly increased enrichment of methyltransferases in comparison with normal breast epithelial cells. Compared to the treatment with DMSO, the treatment with M.SssI induced an increase in the enrichment of methyltransferases, while the treatment with 5-aza-dc caused a reduction in methyltransferase enrichment (p < 0.05) (Figure 3D). According to the results from Western blot analysis, reduced Cosmc levels and elevated expression of the Tn and STn antigens were found in MDA-MB-231 and MCF-7 cells. Compared with DMSO treatment, the treatment with M.SssI caused a reduction in the Cosmc levels and an elevation of the Tn and STn antigens. These changes were reversed following treatment with 5-aza-dc (p < 0.05) (Figure 3E and F). Consequently, these results suggested that the demethylation of Cosmc upregulated the Cosmc levels and inhibited the expression of the Tn and STn antigens.

Figure 3

Demethylation of the Cosmc promoter inhibits the expression of the Tn and STn antigens through the upregulation of Cosmc. (A) CpG island predicted by the MethPrimer website. MDA-MB-231 and MCF-7 cells were treated with methyltransferase M.SssI or methyltransferase inhibitor 5-aza-dc, with DMSO as control. (B) methylation of the Cosmc promoter region in MDA-MB-231 and MCF-7 cells detected by MSP. (C) methylation of Cosmc promoter region in MDA-MB-231 and MCF-7 cells determined by BSP. (D) enrichment of Dnmt1, Dnmt3a, and Dnmt3b in the Cosmc promoter region of MDA-MB-231 and MCF-7 cells evaluated by ChIP. (E and F) protein bands and expression of Cosmc, Tn and STn antigens in MDA-MB-231 and MCF-7 cells assessed by Western blot analysis. *p<0.05 vs normal breast epithelial cell line; #p<0.05 vs MDA-MB-231 and MCF-7 cells treated with DMSO. The above were measurement data and were expressed as mean ± standard deviation. Comparisons between two groups were performed using an independent sample t-test. All experiments were repeated 3 times.

Abbreviations: STn, Sialyl-Tn; DMSO, dimethyl sulfoxide; MSP, methylation specific polymerase chain reaction; ChIP, chromatin immunoprecipitation.

Cosmc Elevation Represses Tumor Growth and Enhances the Apoptosis of Breast Cancer Cells in vivo

To further investigate the effects of Cosmc on breast cancer cell growth in vivo, stably transfected MDA-MB-231 cells were inoculated into nude mice followed by measurement of tumor size and weight in nude mice. The delivery of oe-Cosmc resulted in significantly decreased tumor size and weight (p < 0.05) (Figure 4A). Western blot analysis was conducted to determine the expression of Tn, STn, proliferating-associated proteins (Ki67 and PCNA) and apoptosis-related proteins (Bcl-2, Bax and Bad). The results showed that the delivery of oe-Cosmc contributed to reduced expression of Tn, STn, Bcl-2, Ki67, and PCNA and increased expression of Bax and Bad (p < 0.05) (Figure 4B-C). Therefore, our in vivo findings confirmed that restoration of Cosmc inhibited tumor growth and accelerated apoptosis of breast cancer cells in vivo.

Figure 4

Cosmc overexpression facilitates tumor growth in vivo. Nude mice received an injection of MDA-MB-231 cells stably transfected with oe-Cosmc to restore the expression of Cosmc. (A) tumor size, volume, and weight in nude mice. (B) protein bands and expression of Tn and STn antigens measured by Western blot analysis. (C) protein bands and expression of Ki67, PCNA, Bcl-2, Bax and Bad determined by Western blot analysis. *p<0.05 vs the delivery of oe-NC treated MDA-MB-231 cells. The above were measurement data and were expressed as mean ± standard deviation. n=12. Comparisons between two groups were performed using an independent sample t-test, and comparisons of tumor size at various time points are analyzed by repeated-measures analysis of variance.

Abbreviations: STn, Sialyl-Tn; PCNA, proliferating cell nuclear antigen; Bcl-2, B-cell lymphoma-2; Bax, Bcl2 associated X; Bad, Bcl-2 associated agonist of cell death; NC, negative control.

Discussion

Breast cancer is one of the most common cancers and the main cancer-associated death causes with high occurrence and mortality among women.19 Cosmc, a type of molecular chaperone, interacts with T-synthase to facilitate the synthesis of the T antigen and plays a role in cancer progression.20,21 Recently, DNA methylation offers promising chances for constructing a more comprehensive molecular architecture of breast cancer.22 Therefore, our study aimed to explore the regulatory roles of Cosmc promoter methylation in breast cancer progression. Collectively, the data from this study revealed that the demethylation of the Cosmc promoter inhibited breast cancer progression through suppression of the expression of the Tn and STn antigens.

The initial finding of our study revealed that Cosmc is lowly expressed in breast cancer tissues and cells. Our study showed that Cosmc overexpression decreased levels of Bcl-2, Ki67, and PCNA and elevated levels of Bax and Bad, thus inducing the inhibition of cell growth as well as promoting apoptosis in breast cancer. The Bcl-2 family members can form an interaction network among proteins that play regulatory roles in apoptosis through the permeabilization of the outer membrane of the mitochondria.23 Bcl-2, an anti-apoptotic member of the Bcl-2 family, is regarded as an oncogene capable of inducing various types of cancer.24 Bax is a protein encoded in higher eukaryotes that control the death of cells by apoptosis.25 Bad is an apoptotic protein from the Bcl-2 family and its inhibition plays an inhibitory role in the survival of breast cancer stem cells.26 PCNA is required for reliable DNA replication, which prepares a molecular platform for a great number of protein-DNA and protein-protein interactions at the replication fork.27 Ki67 acts as a prognostic factor to have a potential impact on prognosis prediction in patients with breast cancer after neoadjuvant chemotherapy.28 Consistently, an in vivo nude mice model in this study further confirmed that Cosmc overexpression inhibited tumor formation and promoted cell apoptosis.

Cosmc overexpression increased the expression of Cosmc and T-synthase while decreasing the expression of the Tn and STn antigens. Moreover, suppression of Cosmc at the transcriptional level has been detected in human cancers, and its elevated expression exerts an inhibitory effect on tumor growth by repressing the expression of the Tn and STn antigens.29 The Tn and STn antigens belong to the tumor-associated carbohydrate antigens capable of controlling the interaction among carbohydrates.30 Overexpression of the Tn antigen has been detected in breast cancer tissues, which is correlated with a poor prognosis for overall survival and progression-free survival of breast cancer.31 Moreover, the expression of STn has initially been found at the luminal or apical surface of tissues that appears in the early stages of tumors and has been identified as a potential therapeutic target and a diagnosis and prognosis marker for cancer therapy.14 Also, STn has been revealed to be expressed in breast cancer cells and its expression leads to resistance to chemotherapy and adverse prognosis in breast cancer.8 Thus, loss of Cosmc increases the expression of the Tn and STn antigens, which may contribute to the progression of breast cancer.

Lastly, our experiments confirmed that Cosmc promoter methylation was induced in breast cancer cells. In our current work, we provided evidence that Cosmc promoter methylation downregulated the Cosmc levels, resulting in the expression of the Tn and STn antigens. Hypermethylation of the gene promoter regions can cause the malfunction of genes in multiple human cancers when accompanied by the absence of heterozygosity of the same locus.32 Also, abnormal gene-specific promoter hypermethylation is closely associated with the occurrence and development of breast cancer.33 The absence of Cosmc contributes to the expression of the Tn antigen through the regulation of T-synthase, which is related to the development and progression of various human diseases, including cancer.34,35 For instance, a recent study has found that Cosmc deficient pancreatic cancer cell line tends to induce the expression of the Tn antigen.36 Sun et al have further proved that Cosmc promoter hypermethylation induces the loss of Cosmc and consequent Tn expression in colorectal cancer cell lines.37

Conclusions

Taken together, Cosmc promoter hypermethylation caused the downregulation of Cosmc and promoted the expression of the Tn and STn antigens, which may contribute to the progression of breast cancer (Figure 5). These findings underscore the carcinogenic roles of Cosmc promoter hypermethylation in breast cancer. This study also substantiates that the restoration of Cosmc could be achieved by demethylation, and the anti-tumor effects of Cosmc overexpression have been corroborated both in vitro and in vivo, suggesting Cosmc demethylation as a promising therapeutic strategy for cancer treatment. However, a more comprehensive conclusion about the effects of Cosmc promoter hypermethylation on breast cancer cannot be made due to the limited information available. Therefore, these regulatory mechanisms should be monitored rigorously and reported appropriately in future clinical trials.

Figure 5

The molecular mechanism involving the regulatory roles of Cosmc/Tn/STn in breast cancer. Hypermethylation of the Cosmc promoter induced Tn and STn antigen exposure to promote the occurrence and progression of breast cancer by inhibiting Cosmc levels, while demethylation of Cosmc led to opposite results.

Abbreviation: STn, Sialyl-Tn.