Introduction
As the most common malignancy in women, breast cancer accounts for 18% of female cancer cases [
1]. Each year, there are approximately 1.7 million newly diagnosed breast cancer cases worldwide and 500,000 patients die of breast cancer [
2,
3]. Most breast cancers are invasive. Although distant metastasis in patients diagnosed with early breast cancer accounts for only 5–10% of all cases, the risk of metastasis remains quite high after primary surgical resection and adjuvant radiotherapy. Metastasis of breast cancer increases the cost of treatment and creates an economic burden for patients. Therefore, there is an urgent need to identify the metastasis-associated proteins in breast cancer.
Adenosine triphosphate synthase beta subunit (ATP5B) is the β subunit of F
1F
0-ATP synthase and contains the catalytic site for ATP synthesis as well as hydrolysis [
4]. As a subunit of F
1F
0-ATP synthase, ATP5B was once thought to be strictly located at the inner side of the mitochondrial membrane. However, more recent studies have revealed its ectopic expression on the outer surface of the plasma membrane of highly metabolic cells, where it is called ectopic ATP5B [
5]. Ectopic ATP5B was identified as the receptor for apolipoprotein A on hepatocytes, and it participates in the endocytosis of holo-HDL particles [
6]. In adipocytes, the ectopic ATP5B level is significantly increased during adipogenesis [
7,
8]. Additionally, ectopic ATP5B exhibits antiangiogenic and antitumorigenic effects [
9‐
13]. Antibodies against the β subunit of ATPase show angiostatin-like properties and are cytotoxic to tumor cells by regulating intracellular pH [
14]. Many of the examined markers were of greater or lesser clinical significance [
15‐
18].
ATP5B is transferred to the plasma membrane in a punctate form, similar to the distribution pattern of caveolae. Caveolae are 50–100 nm wide, flask-shaped plasma membrane invaginations that constitute a subcompartment of the cell membrane [
19]. Caveolae serve as microdomains in which signaling molecules and trafficking vesicles are concentrated [
4]. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae and the major structural protein of caveolae, is essential for the formation of caveolae and is implicated in numerous signal transduction pathways [
20]. Cav-1 is an essential role of invadopodia-mediated breast cancer cell invasion by regulating MMP [
21]. Two isoforms of Cav-1 have been found as a consequence of alternative translation initiation sites. Cav-1α has 178 amino acids, and Cav-1β lacks the 32 N-terminal residues of Cav-1α [
22]. However, the functional differences between the two isoforms are still unclear.
In our preliminary work, ectopic ATP5B was identified as a binding partner of metastasis-related short peptide B04 on PC-3 M cells and found to be associated with prostate cancer metastasis [
23]. Furthermore, ectopic ATP5B has a tendency to be highly expressed on the plasma membrane of highly invasive cells, including MDA-MB-231 breast cancer cells. The present study intended to investigate the role of plasma membrane ATP5B in breast cancer migration and invasion and examine the involvement of Cav-1.
Materials and methods
Cell culture
The breast cancer cell line MDA-MB-231 (American Type Culture Collection, Manassas, VA, USA) was routinely cultured in H-DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Double immunofluorescence staining
Cells were fixed first with 2.5% glutaraldehyde at room temperature for 15 min and then with 4% paraformaldehyde for 15 min. After washing in PBS three times, cells were incubated with 5% BSA for 1 h and were then incubated with the primary anti-ATP5B antibody (diluted 1:100) and anti-Cav-1 antibody (diluted 1:200) at 4 °C overnight. After three washes with PBS, cells were incubated with secondary antibodies (anti-mouse IgG Alexa Fluor 488, 1:500; goat anti-rabbit IgM/Alexa Fluor 555, 1:200) for 1 h. Cells were then washed with PBS and stained with Hoechst 33,342 (diluted 1:1000) for 8 min. Images were acquired under an inverted phase contrast fluorescence microscope. The images were acquired using an Olympus FV-1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Construction of Cav-1-shRNA plasmids and stable transfection of cells
All Cav-1-shRNAs were synthesized by GENECHEM (Shanghai, China). The Cav-1 shRNA1 target sequence was 5’- GAC CCA CTC TTT GAA GCT GTT-3’. The Cav-1 shRNA2 target sequence was 5’- CCA CCT TCA CTG TGA CGA AAT-3’. The scrambled shRNA target sequence was 5’- TTC TCC GAA CGT GTC ACGT-3’. Each pair of shRNAs was ligated into the GV418 plasmid. The shRNA fragments within the vector construct were sequenced using the primer H1-F (5’-GGA AAG AAT AGT AGA CAT AAT AGC-3’). The targeting and scramble vectors were transfected into MDA-MB-231 cells using Lipofectamine 2000 reagent (Invitrogen, USA). Briefly, MDA-MB-231 cells were subcultured in 24-well plates (5 × 104 cells/well) at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. The diluted plasmids and liposomes were incubated in serum- and antibiotic-free Opti-MEM for 5 min and were then added to the cell culture plates. The transfected cells were cultured for 6 h and were then transferred to a fresh medium containing 10% FBS. Puromycin was used to select the positive clones.
Immuno precipitation
Plasma membrane proteins were extracted using a Minute™ Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, USA) according to the manufacturer’s instructions. Plasma membrane proteins were precleared with Protein A/G Plus agarose beads, and aliquots of the supernatant were incubated first with the anti-ATP5B antibody or normal mouse IgG overnight at 4 °C and then with Protein A/G Plus agarose beads at 4 °C for 3 h. The beads were collected by centrifugation at 10,000 × g for 15 s at 4 °C and washed with PBS.
Western blotting
Western blotting was performed as described previously [
24]. Briefly, cell lysates were analyzed by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 h at room temperature in buffer containing 10% skim milk powder and were then incubated overnight at 4 °C with different primary antibodies (anti-ATP5B, anti-Cav-1, anti-Na
+-K
+ ATPase α). After washing three times, membranes were incubated with secondary antibodies for 1 h at room temperature. The signals of the detected proteins were visualized with an ECL detection system (Amersham Biosciences, USA).
RT-PCR
Total RNA was extracted using TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s instructions. mRNA levels were assessed with RT-PCR (Transgen, Beijing, China). Gene expression levels were normalized to that of an endogenous reference gene, β-actin. Experiments were performed in triplicate. The primer sequences for β-actin detection were as follows: forward, 5’- TCA TGA AGT GTG ACG TGG ACA TC -3’ and reverse, 5’- CAG GAG GAG CAA TGA TCT TGA TCT -3’. The primer sequences for Cav-1 detection were as follows: forward, 5’- CTA CAA GCC CAA CAA CAA GGC -3’ and reverse, 5’- AGG AAG CTC TTG ATG CAC GGT -3’.
Migration assay
Migration assays were performed in 24-well plates with 8-μm pore-sized chamber inserts (Costar, Corning, Inc.). Approximately 4 × 104 cells/well were resuspended in 200 μl of serum-free medium with or without cholesterol/B04 and were then seeded in the upper chambers. In addition, 700 μl of medium supplemented with 10% FBS was added to the lower chambers. The cells were incubated at 37 °C and 5% CO2 for 24 h, after which the cells on the upper surface of the filters were gently removed by wiping with a moist cotton swab. The cells on the lower surface of the membranes were fixed with paraformaldehyde solution for 10 min and stained with 0.1% crystal violet. The number of migration cells was counted under an inverted phase contrast microscope.
Invasion assay
Invasion assays were performed in 24-well plates by inserting with 8 μm pore size (Costar, Corning, Inc.). Cells were cultured in serum-free medium for 24 h before the invasion assay. The upper chamber inserts were coated with 60 μl of Matrigel (dilution 1:4, BD), and 1 × 105 cells were seeded in the upper chamber of each well. Chamber was incubated for 72 h at 37 °C in a 5% CO2 atmosphere and then matrigel on the upper surface of membrane were removed, and the cells on the lower surface of the membrane were washed and fixed in methanol for 5 min. The number of invaded cells was estimated by staining with 0.1% crystal violet.
Discussion
Metastasis accounts for the vast majority of cancer-related deaths. Migration and invasion of cancer cells is the initial step in tumor metastasis [
26]. It is imperative to identify metastasis-associated proteins. In the past decade, the beta subunit of ATPase has been shown to be ectopically expressed on the outer surface of both normal and tumor cells [
23,
27‐
29] and has been identified as a potential therapeutic target in NSCLC [
30] and malignant paragangliomas [
31]. Ectopic ATP5B is involved in the metabolism of lipids, activation of endothelial cells [
32], development of an acidic tumor microenvironment [
33], and proliferation and apoptosis of cancer cells [
34]. ATP5B located on the inner membrane of mitochondria is essential for the conversion of ADP to ATP and provides energy for various life processes. Ectopic ATP5B expressed on the plasma membrane of metabolically active tumor cells can also synthesize ATP [
28]. In addition, ectopic ATP5B has a tendency to appear on invasive tumor cells. In the preceding work, we confirmed that ATP5B is ectopically expressed on the surface of the highly metastatic human prostate cancer cell line PC-3 M and facilitates the metastasis of PC-3 M cells. The level of ectopic ATP5B in the highly invasive breast cancer cell line MDA-MB-231 is higher than that in the less-invasive cell line MCF-7, suggesting the association of ectopic ATP5B with the migration and invasion of breast cancer cells. We attempted to shed light on the effects of ectopic ATP5B on breast cancer migration and invasion by inducing ectopic expression of ATP5B.
Previously, cholesterol was shown to increase ectopic expression of ATP5B in vascular endothelial cells [
25], whereas consumption of cholesterol by β-cyclodextrin decreases ectopic ATP5B expression. Cholesterol replacement caused by endogenous and exogenous ceramides results in a reduction in the level of plasma membrane ATP5B on Schwann cells [
35], indicating that cholesterol can regulate ectopic ATP5B expression. To induce ectopic ATP5B expression, MDA-MB-231 cells were exposed to cholesterol for various durations. The level of ectopic ATP5B was significantly increased in cells treated with cholesterol for 12 h but not in cells exposed to cholesterol for 24 h. Induction of increased ectopic expression of ATP5B in breast cancer by cholesterol has a time limitation. Plasma membrane ATP5B assists the endocytosis of HDL as the receptor for apoA. When stimulated by extracellular cholesterol, cells may accelerate cholesterol transport by inducing ectopic expression of ATP5B. However, long-term cholesterol loading triggers regulatory mechanisms in cells, and the level of ectopic ATP5B is subsequently restored. B04, a specific inhibitor of ATP5B, inhibited the migration and invasion of MDA-MB-231 cells regardless of cholesterol exposure. The influence of cholesterol on cell migration and invasion was excluded, which means that an increase in the Cav-1 level alone cannot affect cell migration and invasion. Thus, cholesterol promotes the migration and invasion of MDA-MB-231 breast cancer cells by increasing the level of ectopic ATP5B.
Caveolae are cholesterol-rich microdomains, and cholesterol loading is related to ectopic expression of ATP5B, suggesting that ATP5B is ectopically expressed in caveolae. Researchers confirmed this hypothesis via detergent isolation and proteomic analysis [
36,
37]. Caveolae act as molecular hubs, recruiting a large number of signaling molecules. Cav-1 is essential for the formation of caveolae. Lisanti et al. [
38] proposed that Cav-1 participated in the integration of multiple signaling pathways. Like ectopic ATP5B, Cav-1 is expressed on the surface of adipocytes, endothelial cells, epithelial cells, and hepatocytes. Therefore, we speculated that Cav-1 may affect the function of ectopic ATP5B by binding to it. The colocalization of ectopic ATP5B and Cav-1 was confirmed by double immunofluorescence staining. The interaction between ectopic ATP5B and Cav-1 was studied by immunoprecipitation. Proteins immunoprecipitated with an anti-ATP5B antibody were separated by SDS-PAGE for detection of Cav-1. Interestingly, only the shorter variant (21 kD) of Cav-1β was detected. Ectopic ATP5B binds to Cav-1β on MDA-MB-231 cells. Two subtypes of Cav-1 have been described. Cav-1β lacks the first 31 N-terminal amino acids of Cav-1α (residues 1–178). Cav-1β was discovered to be present mainly at the edge of migrating cells [
39]. This study provides further insight into the two variants.
Cav-1 is related to the processes of proliferation, apoptosis, metastasis, and autophagy in tumor cells and participates in the occurrence and development of tumors [
40]. However, the role of Cav-1 in tumors remains controversial [
41]. Its tumor suppressive and oncogenic properties have been reported in different tumors. Intriguingly, both contradictory functions can occur within the same tumor type. The effect of Cav-1 on MDA-MB-231 cells was investigated. Cav-1 enhanced migration and invasion of melanoma and breast cancer cells [
42,
43]. Cav-1 promotes migration of metastatic breast cancer cells [
44]. Interestingly, overexpression of Cav-1 promoted secretion of MMP-3 and MMP-11 (active) proteins, and upregulation of Cav-1 expression in nasopharyngeal carcinoma enhanced tumor cell migration and correlated with poor prognosis of the patients [
45].
The expression of plasma membrane Cav-1 was significantly decreased after knockdown of Cav-1. In addition, the interaction between ectopic ATP5B and Cav-1 was undetected. The possible reason is that the binding of ATP5B and Cav-1 depends on the levels of the two proteins. MDA-MB-231 cells with stable knockdown of Cav-1 showed decreased migration and invasion potential, which was not reversed by cholesterol, indicating that an increase in the level of ATP5B alone cannot affect cell migration and invasion. Cav-1 was indispensable for the migration- and invasion-promoting function of ectopic ATP5B.
This study provides evidence that Cav-1 is critical for ectopic ATP5B expression. Ectopic ATP5B promotes the migration and invasion of MDA-MB-231 cells by binding to Cav-1. The involved downstream signaling pathways need to be explored further in future studies.
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