Background
The tetraspanin family is a group of cell surface proteins that are characterized by four transmembrane domains [
1]. It is well known that tetraspanin proteins regulate several types of physiological properties, including cell morphology, motility, invasion, fusion and signaling of tumors, among others [
2]. The CD63 gene, which is located on human chromosome 12q13, was the first tetraspanin to be characterized [
3]. Recent studies have demonstrated that CD63 interacts with many different proteins, either directly or indirectly, and regulates intracellular transport and localization [
4,
5]. In addition, an increasing number of studies have indicated that the cell surface expression of CD63 is tightly regulated by glycosylation [
6]. In fact, the molecular weight of CD63 has been observed to be 32, 35, or 50 kDa with N-linked glycosylation in western blotting experiments, although the predicted molecular weight of CD63 is 25 kDa [
7]. Furthermore, it has been reported that CD63 is associated with the biological behavior of solid tumors, especially those with metastatic potential [
8]. However, the contribution of glycosylation of CD63 to cancer malignancy is poorly understood.
Previously, we established that glycosylation in multidrug resistance protein 1 (MDR1, also known as ABCB1) is regulated by ribophorin II (RPN2), which is part of an N-oligosaccharyl transferase complex [
9]. RPN2 silencing induced docetaxel-dependent apoptosis and cell growth inhibition of human breast cancer cells through the reduction of P-glycoprotein glycosylation. In addition,
in vivo delivery of RPN2 siRNA inhibited tumor growth in mice given docetaxel. These observations indicated that RPN2 is a key regulator of N-glycosylation in drug-resistant cancer cells. However, little is currently known regarding the association between RPN2 and specific glycosylated proteins that are related to cancer malignancy. In this study, we demonstrate that RPN2 promotes cancer cell malignancy in breast cancer cells through the regulation of CD63 glycosylation.
Discussion
Previous studies have shown that the localization of MDR1 at the cell membrane is important to drug resistance in cancer cells [
12,
13]. Thus, targeting of MDR1 by small molecule compounds or antibodies is an effective strategy for overcoming multiple-drug resistance in cancer [
14]. The ultimate goal of restoring drug sensitivity has been met with limited success in clinical trials thus far, although promising studies on the pharmacological inhibition of MDR1 have indicated that it is possible to sensitize drug-resistant cells. Recently, we determined that RPN2 efficiently induced apoptosis in docetaxel-resistant human breast cancer cells [
9]. In addition, silencing of RPN2 reduced the glycosylation of MDR1 and decreased its membrane localization, thereby sensitizing cancer cells to docetaxel. Our current results indicate that CD63 glycosylation by RPN2 is important for the localization of CD63 and MDR1 in human breast cancer cells. Indeed, Yoshida et al. showed that CD63 had three N-linked glycosylation sites [
15], that CD63 interacts with CXCR4 through the N-linked glycans-portion of the CD63 protein and that the complex induces the direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. Moreover, several reports have shown that CD63 interacts with many different proteins, including integrins and the Src family tyrosine kinases Lyn and Hck, which are known to promote cancer malignancy [
16‐
18]. In addition, recent studies have shown that multi-drug resistant cancer cells overexpressing MDR1 displayed increased invasive activity and metastatic behavior [
19]. These phenotypes were similar to those observed in RPN2-mediated cancer malignancy [
11]. Taken together, our results support the possibility that glycosylated CD63 by RPN2 plays an important role in cancer malignancy through the regulation of protein localization.
We revealed that CD63 interacts with MDR1 and regulates the drug resistance and invasiveness involved in cancer cell malignancy. However, a correlation between decreased expression of CD63 and increased malignancy has also been observed in many other tumors. For instance, it has been reported that CD63 is strongly expressed on the cell surface during the early stage of malignant melanoma, but this localization is weaker or absent in the malignant stages of melanoma compared to normal melanocytes [
8]. By contrast, Huang et al. reported that nearly all breast cancers were positive for CD63 mRNA expression [
20]. As shown in this study, we found that CD63 localization regulated by RPN2-mediated glycosylation is important for malignant characteristics, including drug resistance and invasiveness. Therefore, to evaluate whether CD63 was associated with cancer malignancy, it was essential to evaluate both the glycosylation and expression level of CD63. Furthermore, we demonstrated that CD63 and MDR1 co-localization was associated with LN metastasis in clinical samples, which suggested that co-localization of CD63 and MDR1 is likely to be a cause for treatment resistance in breast cancer patients.
Methods
Reagents
The antibiotic solution (containing 10,000 U/mL penicillin and 10 mg/mL streptomycin), trypsin-EDTA mixture (containing 0.05% trypsin and EDTA), FBS (fetal bovine serum), donkey anti-mouse-Alexa 488, goat anti-rabbit-Alexa 488, and goat anti-mouse-Alexa 594 were obtained from Invitrogen (Carlsbad, CA, USA). Rabbit polyclonal anti-MDR (H-241, sc-8313) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-actin, clone C4 (MAB1501) was purchased from Millipore (Billerica, MA, USA). The mouse monoclonal anti-CD63 monoclonal (H5C6) antibody was obtained from BD Pharmingen (San Diego, CA, USA). Hoechst 33258 dye was obtained from Dojindo (Kumamoto, Japan). Antigen activation of the tissue microarray was achieved using a protease (#415231, Nichirei, Japan). The duplexes of each siRNA targeting human CD63 mRNA (si CD63-1, GGUGGAAGGAGGAAUGAAAdTdT, UUUCAUUCCUCCUUCCACCdTdT; CD63-2, GGCAGCAGAUGGAGAAUUAdTdT, UAAUUCUCCAUCUGCUGCCdTdT; CD63-3, GUGGCUACGAGGUGAUGUAdTdT, UACAUCACCUCGUAGCCACdTdT) were purchased from BONAC Corporation (Fukuoka, Japan). The siRNA duplexes targeting human RPN2 mRNA (GGCCACUGUUAAACUAGAACA, UUCUAGUUUAACAGUGGCCUG) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the AllStars Negative Control siRNA was obtained from Qiagen (Valencia, CA, USA).
Cell culture
MDA-MB-231-luc-D3H2LN (MM231-LN) cells were purchased from Xenogen (Alameda, CA), and multidrug-resistant MCF7-ADR cells were provided by Shien-Lab, Medical Oncology, National Cancer Center Hospital of Japan. These cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic at 37°C in 5% CO2.
Transient transfection assays
Transfection of siRNA was accomplished using DharmaFECT transfection reagent (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. AllStars Negative Control siRNA was used as the negative control (N.C.).
Cell proliferation assay (MTS assay)
Five thousand cells per well were seeded in 96-well plates. The following day, the cells were transfected with siRNAs. After 1, 2 or 3 days of culture, cell viability was measured using a cell counting Kit-8 (Dojindo, Kumamoto, Japan) according to the instructions of the manufacturer. The absorbance at 450 nm was measured using an Envision multilabel plate reader (Wallac, Turku, Finland).
Apoptotic activity
MM231-LN and MCF7-ADR cells (2 × 105 cells in a 6-well plate) were transfected with control, RPN2 or CD63 siRNA as described above. After 2 days in culture, cells were collected, and proteins were extracted with M-PER (Thermo Scientific). Caspase-3/7 activity was assessed using an Apo-ONE Homogeneous Caspase-3/7 Assay system (Promega, Wisconsin, USA) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Envision system (Wallac).
Transwell invasion assay
Breast cancer cell invasion was assayed in 24-well Biocoat Matrigel™ invasion chambers (8 μm; BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, after the transfection of siRNA into the cells, 20,000 cells were plated in the upper chamber containing RPMI 1640 medium without FBS on the following day. The lower chambers were filled with RPMI 1640 medium with 10% FBS as a chemoattractant. Twenty-two hours later, the low-invasive cells were removed with a cotton swab. The cells that migrated through the membrane and adhered to the lower surface of the membrane were fixed with methanol and stained with Diff Quick staining solution (Sysmex, Kobe, Japan). For quantification, the cells in four random fields were counted using a microscope. All assays were performed in triplicate, and the invasive values were normalized to the values from cells transfected with the AllStars Negative Control siRNA.
Isolation of mRNAs and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells using a miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The qRT-PCR method has previously been described [
21]. PCR was performed in 96-well plates using a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and all reactions were performed in triplicate. TaqMan® qRT-PCR kits and human-CD63 and human-β-actin TaqMan® Expression Assays were purchased from Applied Biosystems (Foster City, CA, USA). Reverse transcription (Applied Biosystems, Foster City, CA, USA) and TaqMan® quantitative PCR (Applied Biosystems, Foster City, CA, USA) were performed according to the manufacturer’s instructions. SYBR® Green I qRT-PCR was performed, and the β-actin housekeeping gene was used to normalize the variation in the cDNA levels. The primer sequences are as follows (shown 5′ to 3′): human β-actin, GGCACCACCATGTACCCTG (Forward) and CACGGAGTACTTGCGCTCAG (Reverse); and human RPN2, ATCTAACCTTGATCCCAGCAATUGTG (Forward) and CTGCCAGAAGCAGATCTTTGGTC (Reverse).
Immunoblot analysis
SDS-PAGE gels were calibrated using Precision Plus protein standards (161–0375) (Bio-Rad, Hercules, CA, USA), and anti-CD63 (1:200) and anti-actin (1:1,000) were used as the primary antibodies. The dilution ratio of each antibody is indicated in parentheses. A peroxidase-labeled anti-mouse secondary antibody was used at a dilution of 1:10,000. The bound antibodies were visualized using chemiluminescence with an ECL Plus Western blotting detection system (GE HealthCare, Piscataway, NJ, USA), and luminescent images were captured using a LuminoImager (LAS-3000; FujiFilm Inc., Tokyo, Japan).
Immunofluorescent staining
After being washed three times with PBS, the cells were fixed in 4% paraformaldehyde (Wako, Japan) and incubated in 0.1% BSA containing primary antibodies (anti-CD63 (1:500) and anti-MDR (1:500) for 1 hour. The cells were then incubated in 0.1% BSA containing Alexa Fluor fluorescent secondary antibodies. Nuclei were visualized with Hoechst 33258 dye (Dojindo, Kumamoto, Japan). All staining was observed using a confocal microscope (FluoView FV1000; Olympus, Tokyo, Japan).
Cell membrane labeling
MDA-MB-231-luc-D3H2LN and MCF7-ADR cells were transfected with control or RPN2 siRNA. After 2 days in culture, cells were labeled with a PKH26 red fluorescent labeling kit (Sigma Aldrich). Cells were observed using confocal microscopy (FluoView FV1000; Olympus, Tokyo, Japan). Nuclei were visualized via Hoechst 33258 (Dojindo, Kumamoto, Japan) staining.
Tissue microarrays
The tissue arrays of breast cancer samples (BR1503b, BR10010a) were purchased from Biomax US. The company provided certified documents that all human tissue samples were collected with informed consent from the donors or their relatives. Detailed information on all tumor samples can be found at
http://www.biomax.us/. The tissue microarrays were incubated in 0.1% BSA containing primary antibodies, including anti-CD63 (1:500) and anti-MDR (1:500), for 1 hour after a 5-min protease treatment (Nichirei, Tokyo, Japan). The cells were then incubated in 0.1% BSA containing Alexa Fluor fluorescent secondary antibodies. Nuclei were visualized using Hoechst 33258 (Dojindo, Kumamoto, Japan) staining for observation using a confocal microscope (BZ-9000; Keyence, Tokyo, Japan).
Statistical analysis
The data presented in bar graphs are the mean and s.e.m. of at least three independent experiments. Statistical analyses were performed using Student’s t-test. Associations between lymph node metastasis or MDR expression and CD63 expression were assessed by means of a chi-square test. The statistical analysis was two-sided, and P < 0.05 was considered to be significant.
Acknowledgements
This work was supported, in part, by a Grant-in-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control; a Grant-in-Aid for Scientific Research on Priority Areas of Cancer from the Ministry of Education, Culture, Sports, Science and Technology; the National Cancer Center Research and Development Fund; the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NiBio); a Grant-in-Aid for JSPS fellows; Project for Development of Innovative Research on Cancer Therapeutics; and the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)” that was initiated by the Council for Science and Technology Policy (CSTP). We thank Ms. Ayako Inoue for excellent technical assistance.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NT and KH performed the experimental work, conducted data analysis and wrote the manuscript. NT’s main contribution was obtaining proof of the relationship between CD63 and anti-cancer drug resistance. KH obtained evidence of the relationship between CD63 and invasiveness. NK assisted in the writing of the manuscript and provided helpful discussion. HN provided helpful discussion. TO supervised this project. The manuscript was finalized by TO with the assistance of all of the authors. All authors read and approved the final manuscript.