Background
Breast cancer is a leading cause of cancer-related death in women worldwide. Despite the significant progress that has been made in cancer treatment and early detection, approximately 30 to 70% of breast cancer patients die from breast cancer recurrence or metastasis [
1]. Therefore it is important to investigate the mechanisms underlying cancer cell proliferation and metastasis-related cellular activities such as cell migration and invasion. Genetic mutations or epigenetic modifications that provide growth advantage are critical for tumour initiation and progression. In breast cancer, activation mutations of genes such as
EGFR,
HER2 have been found to transmit signals into the nucleus to up-regulate the transcription of downstream genes, promoting cancer cell growth [
2‐
4]. However, little is known about the mediators in the nucleus that are involved in gene regulation. Interestingly, the transcription factor SOX2 has recently been associated with breast cancer formation and metastasis [
5]. SOX2 belongs to the sex determining region Y (SRY)-like box (SOX) gene family, members of which are pivotal for normal development and maintenance of stemness [
6‐
11], cell proliferation and differentiation [
12]. Emerging studies demonstrated that the SOX2 protein is also involved in tumourigenesis [
13‐
15]. High levels of SOX2 closely correlate with multiple processes during tumour development, including initiation [
16], maintenance [
17,
18], invasion and metastasis [
19‐
21]. Accordingly, aberrant
SOX2 expression was detected in multiple types of cancers at different stages. We and others have shown high levels of SOX2 in lung cancer, esophageal squamous cell carcinomas (ESCC) and ovarian cancer [
13,
22‐
26]. Interestingly, high levels of SOX2 proteins have also been found in 19% of breast cancer patients [
5]. Moreover, SOX2 expression has been linked to tamoxifen resistance and relapse in breast cancer treatment [
27]. Nevertheless, the mechanisms by which high levels of SOX2 regulate the progression and metastasis of breast cancer remain largely unexplored.
MicroRNAs (miRNAs) are small, noncoding RNAs that are important for the regulation of gene expressions at the post-transcriptional level. miRNAs are involved in almost all key cellular activities such as proliferation, differentiation and migration [
28]. Dysregulation of miRNAs is also involved in the initiation and progression of human cancers including breast cancer [
29‐
31]. For example, miR-124 is down-regulated in breast cancer cell lines. Ectopic expression of miR-124 reduces the protein levels of Myc and phospho-Rb while up-regulating p27 in the breast cancer cell line MDA-MB-231 [
32], suggesting that miRNAs function as a tumour suppressor. In this study we examined the molecular mechanisms used by SOX2 in the proliferation and migration of breast cancer cell lines and identified that SOX2 regulates two miRNAs (miR-181a-5p and miR30e-5p) which in turn influence the expression of a common downstream target TUSC3. Importantly, inversed expression of SOX2 and TUSC3 is associated with poor prognosis of a subpopulation of breast cancer patients. Therefore we demonstrate here a clinically relevant axis which involves SOX2/miRNAs/TUSC3 in breast cancer development.
Methods
Cell lines and mice
ZR7530 (Cat NO: CRL-1504™) and MDA-MB-231 (Cat NO: HTB-26™) breast cancer cell lines were purchased from ATCC (Manassas, VA, USA), Cell line authentication was conducted by short tandem repeat analysis, and mycoplasma contamination in cell lines was tested using multiplex PCR. They were infected with lentivirus carrying
pLK0.1SOX2 (SOX2 knockdown plasmid),
pCDH-SOX2-IRES-GFP (SOX2 overexpression plasmid),
pLK0.1Scramble (control plasmid) [
33], and
pCDH-TUSC3-IRES-GFP (TUSC3 overexpression plasmid, primers used for construction were shown in Additional file
1: Table S1). Puromycin (Santa Cruz Biotechnology, Cat No: sc-108071, Dallas, TX, USA) selection was used to establish cell lines stably expressing these constructs. Cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, HyClone, Cat No: SH30022.01B, Beijing, China) with 10% foetal bovine serum (FBS, Gibco, Cat No:10270-106, Grand Island, NY, USA). Nude mice at 6-weeks old were purchased from SLRC Laboratory Company (Shanghai, China).
Western blot analysis
Western blot was performed as previously described [
34]. PVDF membrane was used for protein transfer and probed with antibodies against human SOX2 (polyclonal rabbit anti-SOX2, SEVEN HILLS, Cat No: WRAB-1236, Cincinnati, USA), human β-actin (mouse anti-β-actin, Beyotime, Cat No: AA128, Nantong, China), CDK4 (BBI, Cat No: AB20396b, Shanghai, China), CDK6 (BBI, Cat No: AB20398a, Shanghai, China), CCND1 (BBI, Cat No: AB60236b, Shanghai, China), TUSC3 (Novus Biologicals, Cat No: NBP1-55630, Littleton, USA). HRP-conjugated goat anti-rabbit IgG (Abcam, Cat No: ab136817, Cambridge, MA, USA) and HRP-conjugated goat anti-mouse IgG (ZSGB-Bio, Cat No: ZB-2305, Beijing, China) were used as secondary antibodies to detect the proteins. The density of protein band was quantified with Image-Pro Plus 6.0 software, and the ratio of target protein to housekeeping protein, which reflects the change of expression level, was calculated.
Cell proliferation and colony formation assays
ZR7530 or MDA-MB-231 cells with genetic manipulation (knockdown or overexpression) were seeded in 96-well plates. The OD value of cells was measured under 450 nm and 630 nm at 24, 48, 72 and 96 h using the Cell Counting Kit 8 (CCK-8) (Beyotime) assay, and the absorbance difference between 450 nm and 630 nm represents cell proliferation rates. For soft agar colony formation assays, ZR7530 or MDA-MB-231 cells were seeded in 6-well plates and maintained for 2 weeks and colonies were fixed with 4% paraformaldehyde (DingGuo, Cat No: AR-0211, Beijing, China) and stained with 0.5% crystal violet. The number of total colonies and colonies with a diameter greater than 0.5 mm were calculated with Image J (Fiji-win32 software, University of Wisconsin-Madison), and the data were analysed as previously described [
35,
36].
Wound healing assay
1 × 10
6 ZR7530 or MDA-MB-231 cells (SOX2 knockdown, scramble and TUSC3 overexpression) were seeded in 6-well plates and grown to 90% confluence. Cell monolayers were scraped with a sterile pipette tip. Floating cells were removed and the cultures were maintained in DMEM supplemented with 5% FBS. The wound area was recorded 0 h and 48 h after the scrape. The healing index was calculated and analysed using the formula: (S
0 − S
n)/S
0 × 100%, which represents the ability of cellular migration. S
0 represents the blank area 0 h after scraping, and S
n represents the blank area n h after scraping [
37].
Matrigel invasion assay
Invasion assays were performed in chambers with a 6.5-mm insert in 24-well plates (Corning company, Cat No: 3422, Corning, NY, USA). An 8-μm polycarbonate membrane was coated with 12 μl of ice-cold Matrigel™ Basement Membrane Matrix (BD company, Cat No: 356234, Bedford, MA, USA). Cells (5x104 per well) were added to the upper chamber in 200 μl of the medium containing 5% FBS. The lower chamber was filled with 500 μl of medium containing 20% FBS. The culture was maintained for 24 h. Cells were then fixed in methanol and stained with 1% crystal violet. Cells at the lower side of the filter were counted under a light microscope and dissolved with 50% acetic acid.
Xenograft studies
1x10
5 ZR7530 cells were re-suspended in 200 μl of DMEM and subcutaneously injected into the flanks of 6-week old BALB/c nude mice, which were maintained in the SPF environment. Five female nude mice per cohort (SOX2 knockdown and control) were randomly divided to receive cancer cells. Tumour size was measured every 3 days using a Vernier calliper after injection, and tumour volumes were calculated with the formula 0.52 × width
2 × length [
38,
39]. Tumours were collected 7 weeks after cell inoculation.
For lung metastatic experiments, 2 × 10
6 tumour cells were re-suspended in 100 μl of PBS and injected into BALB/c nude mice via the tail vein. Five female nude mice per cohort, which were also maintained in the SPF environment, received the tumour cells through tail vein. These nude mice were sacrificed 8 weeks after tail vein injection. Lungs were fixed with 4% PFA and sectioned for haematoxylin and eosin (HE) staining to determine the metastatic capabilities of cancer cells. The metastatic capabilities were assessed as previously described [
40,
41].
miRNA microarray assay and real-time PCR
Total RNAs of ZR7530 cells (SOX2 knockdown and control) were extracted and purified using Trizol (Invitrogen, Carlsbad, CA, USA) and miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The human miRNA microarray miRCURY™ LNA Expression Array (v.18.0, Exiqon) was used to detect the level changes after SOX2 knockdown. The low intensity miRNAs were filtered, and original signal intensities were normalised using the Median Normalization Method. The miRNA data were analysed with Significance Analysis of Microarrays (SAM) version 2.1 to generate a list of miRNAs that up-regulate or down-regulate with a fold change ≥1.5 and a
p-value ≤0.05. Quantitative real-time PCR analysis was used to validate the level changes of miRNA. Additional file
1: Table S2 and Table S3 describe the stem-loop RT primers and specific PCR primers used for quantitative PCR.
GO and pathway analyses
Differentially expressed miRNAs were clustered using the Cluster software, and we performed miRNA target gene predictions using the microRNA target prediction tools miRBase, TargetScan, and miRanda, and all common target genes which are regulated by two or multiple miRNAs were analyzed with cytoscape program. We also performed Gene Ontology (GO) analysis (
http://www.geneontology.org) of the target genes that were predicted using the three databases, and the KEGG database (
http://www.genome.jp/kegg/) was selected for pathway analysis based on the target genes of these differentially expressed miRNAs. The significance of the GO terms and Pathway was selected based on a
p-value cut-off of 0.05.
Luciferase reporter assays
Luciferase reporter activities were determined using the Dual-Luciferase Reporter Assay System (Promega, Cat No. E1910, Madison, USA). The primers used for obtaining mutated 3’UTR of TUSC3 were described in Additional file
1: Table S4. pGL3 with wild-type or mutated 3’UTR of TUSC3, miRNA mimics and pRL-TK were co-transfected into cells for all luciferase assays, and Renilla luciferase activities were measured to assess the transfection efficiency, sequences of miRNA mimics, inhibitors and relevant controls were shown in Additional file
1: Table S5.
Statistical analysis
The data represent at least three independent experiments using cells or extracts from a minimum of three separate isolations. Differences between groups were compared using analysis of variance for repeated measures. All statistical analyses were performed using GraphPad PRISM. v5.0 software (San Diego, CA, USA). Data were presented as mean ± SD (standard deviation). Statistical significance between two groups was calculated by unpaired student’s t-test, and P value < 0.05 is considered significant.
Discussion
High levels of SOX2 proteins have been linked to breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here, we use in vitro and in vivo models to demonstrate that SOX2 is required for multiple steps in metastatic processes. We showed that SOX2 regulates the proliferation, migration and seeding of breast cancer cells. More importantly, we have identified a miRNA-mediated regulatory axis downstream of SOX2 to control these processes. Specifically, we found that SOX2 regulates the levels of miR-181a-5p and miR-30e-5p which in turn modulate the levels of the tumor suppressor TUSC3. Consistently, overexpression of TUSC3 reduces the proliferation and migration of SOX2-high breast cancer cells.
SOX2 is a key transcription factor important for the self-renewal of stem cells in multiple tissues including the trachea and brain [
6]. We and others have shown that SOX2 overexpression promotes the proliferation of stem cells in the esophagus and lung, leading to malignancy [
15,
33], suggesting that SOX2 acts as an oncogene. In this study, we found that SOX2 is also required for the proliferation of breast cancer cells. SOX2 knockdown leads to decreased levels of cell cycle proteins including CCND1, CDK4 and CDK6. Interestingly, SOX2 knockdown also leads to reduced levels of multiple microRNAs. We found that the levels of miR-30e-5p (previously named as miR-30e) are most dramatically down-regulated upon SOX2 knockdown, which was validated by real-time PCR. Interestingly, we also found that miR-181a-5p (previously named as miR-181a) which has previously been linked to breast cancer development is also dramatically downregulated. More importantly, further analysis using the cytoscape program we found that miR-30e-5p together with miR-181a-5p co-regulate a common downstream target, TUSC3. Both miR-30e-5p and miR-181a-5p have been implicated in the regulation of cancer cell growth and migration, invasion and metastasis [
52‐
54]. miR-181a-5p is a member of the miR-181 s family which includes three other highly conserved miRNAs, miR-181b, c, and d [
55]. Previous studies have shown increased levels of miR-181a-5p in multiple types of cancer including ovarian cancer and gastric cancer [
56,
57]. The levels of miR-181a-5p in breast cancer were recently assessed. While Li et al. demonstrated that miR-181a-5p is down-regulated in aggressive human breast and colon cancers [
58], a recent study showed that the levels of miR-181a-5p are high in breast cancer samples [
59]. By contrast, we have just begun to elicit the role of miR-30e-5p in tumour development. Tissue microarray analysis suggests that high levels of miR-30e-5p in primary tumours are associated with a favourable prognosis in breast cancer [
60]. In our study, we observed increased levels of miR-181a-5p and miR-30e-5p in breast cancer biopsies as compared to normal mammary gland tissue. We found that SOX2 knockdown leads to decreased levels of miR-181a-5p and miR-30e-5p accompanied by decreased cell proliferation, suggesting that these two microRNAs are required for the proliferation of breast cancer cells.
Consistently, we identified that miR-181a-5p and miR-30e-5p target the tumor suppressor TUSC3. Overexpression of these two miRNAs reduces the protein levels of TUSC3. Moreover, the luciferase assay showed that miR-181a-5p and miR-30e-5p regulate the 3’-UTR of TUSC3 transcripts. TUSC3 is a putative tumour suppressor often lost in epithelial cancers e.g. ovarian cancer and head and neck squamous cell carcinomas [
47,
61]. Promoter methylation leading to silencing of
TUSC3 gene was considered as a poor prognostic factor in ovarian cancer [
48]. Here, we show decreased but not complete loss of TUSC3 protein in breast cancers, suggesting that promoter methylation is unlikely a significant contributor to disease progression. We found that the protein levels of TUSC3 are decreased in breast tumour tissues as compared to surrounding normal tissues. In addition, overexpression of TUSC3 decreases the proliferation and migration capabilities of breast cancer cells. These findings are consistent with the tumor suppressor function of TUSC3 [
62]. TUSC3 is an important component of the oligosaccharyltransferase (OST) complex that catalyzes N-linked glycosylation of proteins in the endoplasmic reticulum (ER) [
63,
64]. Loss of TUSC3 in mammalian cells leads to accumulation of unfolded or misfolded proteins in the ER, resulting in ER stress. In ovarian cancer cells loss of TUSC3 induces ER stress and morphological alterations in the ER. Interestingly, ovarian cancer cells lacking TUSC3 seem able to alleviate the massive ER stress response through the PERK-mediated adaptive pathway [
65], which is consistent with the finding that accumulated unfolded proteins in cancer cells induce the unfolded protein response (UPR), facilitating cancer cells to adapt to ER stress [
66]. miR-181a-5p overexpression has been shown to promote ER stress and leads to myogenic differentiation in C2C12 cells [
67]. Along these lines our study indicates that high levels of TUSC3 expression may induce a cellular response of breast cancer cells, resulting in reduced cell proliferation and migration.
Previous studies revealed that SOX2 protein levels are correlated with the overall survival of patients with multiple types of cancer including esophageal [
33] and lung cancer [
24]. Moreover, SOX2 protein has been assessed as a diagnostic marker and therapeutic target for multiple cancer treatments [
68‐
70]. Our results suggest that high levels of SOX2 protein are associated with poor prognosis of breast cancer.
Acknowledgments
The authors are grateful for inputs from colleagues in Liu and Que laboratory.