Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

Abstract Breast cancer stem cells (BCSCs) have been considered responsible for cancer progression, recurrence, metastasis and drug resistance. However, the mechanisms by which cells acquire self‐renewal and chemoresistance properties are remaining largely unclear. Herein, we evaluated the role of miR‐708 and metformin in BCSCs, and found that the expression of miR‐708 is significantly down‐regulated in BCSCs and tumour tissues, and correlates with chemotherapy response and prognosis. Moreover, miR‐708 markedly inhibits sphere formation, CD44+/CD24− ratio, and tumour initiation and increases chemosensitivity of BCSCs. Mechanistically, miR‐708 directly binds to cluster of differentiation 47 (CD47), and regulates tumour‐associated macrophage‐mediated phagocytosis. On the other hand, CD47 is essential for self‐renewal, tumour initiation and chemoresistance of BCSCs, and correlates with the prognosis of breast cancer patients. In addition, the anti‐type II diabetes drug metformin are found to be involved in the miR‐708/CD47 signalling pathway. Therefore, our study demonstrated that miR‐708 plays an important tumour suppressor role in BCSCs self‐renewal and chemoresistance, and the miR‐708/CD47 regulatory axis may represent a novel therapeutic mechanism of metformin in BCSCs.

| 5995 TAN eT Al. enriched in CD44 + /CD24 − population and are thought to trigger drug resistance and cancer metastasis. 4,5 Previous studies showed that miRNA-708 can suppress cancer progressions in numerous types of cancers. [6][7][8][9][10][11] In breast cancer, miR-708 overexpression inhibited cancer metastasis by targeting neuronatin. 6 Despite these findings showing miR-708 as a potential tumour suppressor, its function in breast cancer stem cells (BCSCs) has not been reported, and how miR-708 is regulated and the detailed mechanism is still unknown. In addition, it is likely that there are additional targets of miR-708 involved in the regulation of BCSCs chemoresistance and cancer metastasis.
Over the past decade, accumulating evidence shows that the blockade of immune checkpoint has attracted wide attention and made great progress in anti-cancer treatment. 12 Macrophage phagocytosis checkpoint has been demonstrated to be an interesting and promising therapeutic target. Cluster of differentiation 47 (CD47) is a ubiquitously expressed cell-surface glycoprotein of the immunoglobulin superfamily that plays a critical role in evasion of immunological eradication. [12][13][14] The overexpression of CD47 in various cancers is clinically correlated with patients' poor prognosis. 15,16 The main mechanism of CD47-mediated immune escape is that it can interact with signal regulatory protein-alpha (SIRPα) on the surface of macrophages to ultimately blocking phagocytosis. [17][18][19] Moreover, CD47 expression is highly increased in CSCs that is mediated by hypoxia-inducible factor 1 and agents targeting CD47 may be developed to eradicate CSCs. 20 Furthermore, we search for the literatures and find CD47 is the predicted target gene of miR-708, which can be predicted by current target gene prediction softwares. 9 In recent studies, it was reported that metformin, a biguanide anti-diabetic drug for the type II diabetes, can selectively reduce the number of BCSCs and suppress tumour development. 21 Moreover, the expression of PD-L1 in tumour cells can be effectively inhibited by metformin. 16 These findings imply that metformin may exert its antitumour effects through regulating immune-related genes and signalling pathways.
Here, we report that miR-708 is significantly down-regulated in BCSCs and inhibits self-renewal and chemoresistance of BCSCs in vitro and in vivo. Interestingly, metformin reduces the stem cells through miR-708 mediated repression of the gene CD47, which is involved in type II diabetes development. Moreover, miR-708 and CD47 regulate the phagocytosis of BCSCs by macrophages. Furthermore, our clinical data suggest miR-708 and CD47 expression in primary breast cancer tissues is associated with patients' prognosis and response to chemotherapy. Overall, the results presented here expound a novel molecular mechanism for miR-708 and CD47 in BCSCs and reveal that metformin plays an important role in this process. Moreover, the results might contribute to a better understanding of the present biological connection between breast cancer and type II diabetes.

| Cell lines and mammosphere culture
Human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from American Type Culture Collection and cultured as previously described. 22 All adherent cells were maintained at 37°C in a 5% CO 2 incubator. For mammosphere culture in vitro, single cells were seeded into a 0.8% agarose-coated 6-well plate at a density of 3,000 cells per well with 2 mL MammoCult TM medium containing 4 μg/mL heparin and 0.48 μg/mL hydrocortisone. Cells were incubated in a 5% CO 2 incubator at 37°C for 10-14 days, and mammospheres were counted under microscope with size >100 μm. For tumour inoculation in vivo, mammosphere cells that were obtained as described above were inoculated into the mammary fat pad of 4-6 weeks old female BALB/c nude mice (obtained from Beijing Vital River Laboratory Animal Technology Company Limited) with or without miR-708 overexpression (CD47 knockdown), and mice were monitored for tumorigenesis in the following one months.
Metformin was used at 0.1-10 mM and docetaxel (Sanofi Aventis) was used at 0-1000 μM. All animal experiments in our study were approved by the Animal Ethics Committee of Sun Yat-sen University and conducted in the Animal Center of Sun Yat-sen University.

| BCSCs marker analysis by flow cytometry
We assessed the expression of CD44 and CD24 surface markers after transfection with miR-708 (or CD47 shRNA). Briefly, cells were washed once in PBS and stained with anti-CD44 (APC-conjugated; BD Biosciences) or anti-CD24 (PE-conjugated; BD Biosciences) antibody in PBS containing 1% FBS and incubated 4°C for 25 minutes.
The cells were washed again with cold PBS and >10 000 cells were used for flow cytometric analysis (FACScalibur, BD Biosciences).

| shRNA, lentiviruses and transduction
All lentiviral vectors in our study have a puromycin resistance gene. To overexpress or knockdown miR-708 in cancer cells, about 500 bp of pri-miRNA containing the mature miR-708 sequence 5′-AAGGAGCUUACAAUCUAGCUGGG-3′ or the miRNA sponge (anti-miR-708) with sequence 5′-CCCAGCTAGATCATAGCTCCTT-3′ was amplified or synthesized, and then cloned into the lentiviral con-

| MiRNA extraction and quantitative realtime PCR
Total RNA and miRNA were isolated from cells and fresh tumour tissues using miReasy micro kit (Qiagen) and from paraffin-embedded normal and malignant breast tissues using RecoverAll Total Nucleic Acid Isolation Kit (Ambion). A total of 500 ng of RNA were reverse transcribed in accordance with manufacturer's instruction (Superscript II reverse transcriptase, Invitrogen). miRNAs expression levels were quantified using TaqMan probes. Expression levels were normalized to those of RNU6B, and relative expression was calculated using the 2 ΔΔCt method.

| Luciferase reporter assays
Wild-type 3′ UTR of CD47 were amplified from genomic DNA by PCR. The mutant CD47 3′ UTR was synthesized using (Invitrogen). The dual luciferase reporter assay was performed 24 hours after transfection using dual luciferase assay system (Promega) according to the manufacturer's protocol. All experiments were performed in triplicate.

| Immunoblot assay
Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP-conjugated secondary antibodies (GE Healthcare) were used, with the anti-actin antibody as an internel control (Santa Cruz).

| Phagocytosis assay
Breast cancer cells were labelled with 5-μM carboxyfluorescein succinimidyl ester (CFSE) and incubated for 15 minutes at 37°C in a CO 2 incubator protected from light according to the manufacturer's protocol (Invitrogen). Macrophages were incubated in serum-free medium for 2 hours before adding 2 × 10 5 CFSE-labelled breast cancer cells. After coculture at 37°C for 2 hours, cells were harvested, macrophages were stained with APC-labelled anti-F4/80 (Novus Biologicals), and phagocytosis were measured and analysed.
Phagocytosis was calculated as the percentage of F4/80 + CFSE+ cells among CSFE + cells.

| Patients and tissue samples
We enrolled 473 patients who underwent primary breast surgery for stage I-III invasive breast carcinoma in the Sun Yat-sen University Cancer Center (SYSUCC). The study was approved by the Institutional Review Board of the SYSUCC and informed consent was obtained from all patients. For survival analyses, relapse free survivals in breast cancer patients were stratified by expression of the miR-708 or CD47 expression and presented as Kaplan-Meier plots. All clinical information of patients used in our study is summarized in Table 1.

| Statistical analysis
Data are expressed as the mean ± SD. Unless otherwise stated, Analyses of statistical significance were performed using the nonparametric Mann-Whitney U test using the GraphPad Prism statistical program. The P values < 0.05 were considered. Error bars depict SD.

| The expression of miR-708 is markedly down-regulated in BCSCs
In many previous studies, CSCs have been shown to regulate the expression of some miRNAs. 23,24 In particular, the miR-708 were reportedly down-regulated in prostate CSCs. 11 To evaluate whether BCSCs derived from MDA-MB-231 and MCF-7 cells had a stronger self-renewal capacity, we performed a sphere formation assay in vitro and a tumour initiation assay in vivo. First, we conducted qRT-PCR analysis of mammospheres, mammospheres re-adhered for 12, Figure 1A

| MiR-708 associates with breast cancer clinical characteristics and good prognosis
To investigate whether miR-708 was involved in clinical breast cancer progression, we detected and analysed miR-708 expression in breast cancer specimens. Remarkably, miR-708 displayed down-regulated in breast cancer tissues compared with normal tissues ( Figure 1E).
Moreover, miR-708 was higher expression in neoadjuvant chemotherapy responders than non-responders, indicating that miR-708 might be associated with chemoresistance ( Figure 1F). Furthermore, in patient samples, miR-708 expression was not associated with conventional clinicopathological parameters (Table 1), however, Kaplan-Meier survival analysis results showed that patients with high miR-708 expression in breast cancers had significantly better overall survival (OS) and disease free survival (DFS) ( Figure 1G,H).
These data demonstrate that miR-708 associates with chemosensitivity of breast cancer and may serve as a marker of good prognosis in breast cancer.

| MiR-708 inhibits the BCSCs
To assess the role of miR-708 in cancer stemness, we evaluate the effect of miR-708 overexpression on the self-renewal capacity of BCSCs using a mammosphere formation assay. As expected, cells with miR-708 overexpression formed fewer mammospheres than cells transfected with negative control (NC) in both MCF-7.SC and MDA-MB-231.SC (Figure 2A). Mammospheres generated from NC groups were larger and grew more rapidly than those with miR-708 overexpression groups. (Figure 2A). In addition, we examined the CD44 + /CD24 − population following transfection with miR-708 or NC. FACS analysis indicated that the overexpression of miR-708 significantly reduced the CD44 + /CD24 − population ( Figure 2B). These results provide evidence that miR-708 can inhibit the self-renewal capacity of BCSCs with high efficiency. Furthermore, in order to address the effect of miR-708 overexpression in BCSCs on tumour formation, we used the limited dilution and tumorigenesis assay in vivo.
As shown in Figure 2C, for both the NC group and the miR-708 overexpression group, 1 × 10 5 MDA-MB-231.SC cells formed tumour xenografts in nude mice with 100% efficiency, but the efficiency in the miR-708 overexpression group is only 75%. When we decreased the cells to 1000 cells, tumours still formed in the NC group with 60% efficiency, but in overexpression group the efficiency decreased to    Figure 2C). Additionally, miR-708 overexpression significantly decreased the tumour weights ( Figure 2D).

| Identification of CD47 as a direct target of miR-708
To identify the target of miR-708 that mediates the generation of stem cells in breast cancer, we searched for predicted target genes using miRanda ( Figure 3A) and found the mirSVR score of CD47 ranked near the top in the obtained list. To further evaluate whether miR-708 affects CD47 expression, we transfected MDA-MB-231.SC and MCF-7.SC with miR-708 and NC. The targeted role of miR-708 on the 3′-UTR of CD47 mRNA was assessed using luciferase assay.
The luciferase activity were decreased in wide type CD47 3′-UTR groups following miR-708 overexpression, while that were not observed in the mutant type groups ( Figure 3B,C). Moreover, we transfected miR-708 into the stem cells and detected the CD47 expression. The mRNA levels of CD47 in the above cell lines were significantly decreased after ectopic miR-708 expression ( Figure 3D).
In addition, an immunoblot analysis was also conducted, and data revealed that CD47 was expressed at a lower level in miR-708 overexpression stem cells (MDA-MB-231.SC miR-708 and MCF-7.SC) than NC stem cells ( Figure 3E). These results show that miR-708 inhibits both mRNA and protein expression of CD47 in BCSCs.

| shCD47 eradicates the BCSCs
To further assess the role of CD47 in BCSCs, we constructed shRNA targeting CD47 (shCD47), and evaluate the effect of knockdown of CD47 expression on the self-renewal capacity of BCSCs. As expected, cells with CD47 knockdown formed fewer mammospheres than cells transfected with NC in both MCF-7.SC and MDA-MB-231.
SC ( Figure 4A). Mammospheres generated from NC groups were larger and grew more rapidly than those from shCD47 groups.
( Figure 4A). In addition, we examined the CD44 + /CD24 − popula- When we decreased the cells to 1000 cells, tumours formation efficiency decreased in both groups, especially in shCD47 group with 20% ( Figure 4C). Furthermore, knockdown of CD47 expression significantly decreased the tumour weights ( Figure 4D). Thus, CD47 loss-of-function contributed to eradicate the BCSCs. Furthermore, considering that miR-708 was demonstrated to be associated with breast cancer clinical characteristics and good prognosis, we then analysed clinical relevance of CD47 in breast cancer. Interestingly, The CD47 level in primary breast tumours was significantly positive correlation with miR-708 expression (Table 1) and patients' poor survival ( Figure 4E,F).

| CD47 deficiency and miR-708 overexpression increase the phagocytosis of macrophages and chemosensitivity in breast cancer
We observed decreased expression of CD47 in stem cells treated with miR-708 ( Figure 3D,E), leading us to hypothesize that miR-708

| MiR-708/CD47 signal pathway is a target of metformin
Previous studies showed that metformin can selectively targets BCSCs and suppress breast tumour growth. 21,25,26 In addition, CD47  Figure 6C,D). These results suggest that CD47 is a novel target of metformin in breast cancer cells and that metformin attenuates stem cells through miR-708-mediated suppression of CD47. BCSCs is a prerequisite for developing more effective cancer treatment. However, whether CSCs also affects the expression of CD47 has not been reported in breast cancer. In our study, we observed that mammosphere cells derived from adherent cells can stimulate the CD47 expression. In addition, CD47 was also identified as a novel target of miR-708 that controls the phagocytosis activity of macrophages by regulating its expression. The results presented here also demonstrate that CD47 regulates the drug resistance of the stem cells in breast cancer. What is more, the expression of CD47 was elevated in breast cancer patients with poor prognosis, then incorporating CD47 into our clinicopathological staging system may be useful in future.

| D ISCUSS I ON
Type 2 diabetes mellitus has been reported to be associated with increased incidence and mortality of a number of malignancies, including breast cancer. [29][30][31][32][33] Previous study results have displayed that metformin selectively blockades the inflammatory pathway in BCSCs by inhibiting signal transducers and activators of nuclear factor-kB, thus inhibiting BCSCs phenotypes. 21,34 However, the molecular mechanism of metformin restoring chemosensitivity of CSCs remains unclear. We stated the fact that metformin regulates miR-708/CD47 axis to eradicate BCSCs and enhance chemosensitivity. It should be noted that we could not elucidate the molecular mechanism and the role of CD47 in the acquisition of tumour seeding ability; therefore, additional investigations of CD47 functions are required. However, the results of our study may provide a new molecular mechanism for the anti-BCSCs effect of metformin.
To sum up, the function of miR-708 in increasing chemosensitivity and attenuating tumour stem cells suggests that RNA-based treatment may improve the outcome of breast cancer patients while routine chemotherapy is performed. Moreover, our study has some implications for raising the existing understanding of biological relationships between breast cancer initiation and type 2 diabetes mellitus development.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
This study was approved the Institutional Review Board of Sun Yatsen university cancer center (GZR2013-108).

CO N S E NT FO R PU B LI C ATI O N
We have received consents from individual patients who have participated in this study. The consent forms will be provided upon request.

ACK N OWLED G M ENTS
We are thankful to the involved patients for its contribution.

CO N FLI C T O F I NTE R E S T S
We declare that we have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets supporting the conclusions of this article are included within the article and its Additional files.