Background
Chemoresistance is a major obstacle for effective breast cancer chemotherapy [
1]. The generation or acquisition of cancer chemoresistance is not clear understood but has been attributed to alterations in many molecular pathways, which include drug metabolizing enzymes and drug transporter genes [
2]. Alterations in the efflux transporters of the ATP-binding cassette (ABC) family have been identified as major determinants of chemoresistance in tumor cells by decreasing the intracellular accumulation of drugs [
3]. In addition, drug-metabolizing enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGTs) have also been reported to constitute a resistance phenotype [
4‐
6]. So far, little is known about the regulation of efflux transporters and metabolizing enzymes, though it has been found to be governed by nuclear receptors [
6].
Spindlin (SPIN), a member of the
SPIN/SSTY family, was first reported as a major maternal transcript expressed in the mouse during the transition from oocyte to embryo [
7]. Recent evidences showed that human Spindlin1 (SPIN1) was highly expressed in ovarian cancer and liposarcoma, and may be implicated in tumorigenesis and development [
8‐
10]. SPIN1 has been shown to be elevated in chemoresistant and metastatic breast cancer tissues and involved in PI3K/Akt-mediated chemoresistance [
11]. SPIN1 has been shown to be a histone code reader to bind histone H3 trimethylated at lysine 4 (H3K4me3) [
12,
13], a chromatin mark typically located at promoters and associated with active or poised genes [
14]. However, whether SPIN1 could regulate the drug metabolizing enzymes and transporters has not been defined. Moreover, we have previously found that SPIN1 was directly regulated by microRNA-489 [
11]. Most microRNAs (miRNAs) only modestly affect their mRNA targets in a fine-tuning manner [
15], and it has been confirmed that multiple miRNAs could target the same gene, suggesting that it is the combination of all these activities that exerts huge impacts on the expression of miRNA target genes [
16]. We hypothesize that SPIN1 may also be regulated by other miRNAs.
Here we show that SPIN1 is highly expressed in drug-resistant breast cancer cells and tissues. We find that SPIN1 increases breast cancer Adriamycin resistance via enhancing the expression of drug metabolizing enzymes and transporter CYP2C8, UGT2B4, UGT2B17 and ABCB4. Mechanistically, the miR-148/152 family could directly target SPIN1 and increase Adriamycin sensitivity in breast cancer cells.
Methods
Tissue samples
We enrolled 78 cases of breast cancer patients before their first neoadjuvant Adriamycin-based chemotherapy cycle, in Qilu Hospital of Shandong University (Jinan, China) between Jul 2008 and Feb 2014. The cases included in this study were invasive breast carcinomas (invasive ductal carcinoma,
n = 73; invasive lobular carcinoma,
n = 5). Pre-chemotherapy needle biopsy samples and the paired surgically removed samples were collected, and the patients were divided into a drug-resistant group and a drug-sensitive group according to the pathological Miller/Payne assessment [
17]. This is a five-point scale that focuses on the principal manifestation of chemotherapeutic effect on reduction in tumor cellularity of resection samples compared with pre-treatment needle biopsy tissues. The tumors were scored blindly by two pathologists and agreement by consensus was achieved if necessary. Patients were divided into a drug-resistant group (Miller/Payne grades 1–2) and a drug-sensitive group (grades 3–5) using the Miller/Payne grading system, as previously described [
18,
19]. This study was approved by the Ethics Committee of School of Medicine, Shandong University (approval code: 2012028).
Immunohistochemistry (IHC)
The streptavidin-peroxidase-biotin (SP) immunohistochemical method was utilized to determine the expression of SPIN1 in breast cancer tissues and xenograft tumors. The paraffin-embedded specimens (4 μm) sections were incubated with the anti-SPIN1 antibody (1:150, Proteintech, 19531-1-AP). For negative controls, the anti-SPIN1 antibody was replaced with PBS. For each sample, 500 cells from five randomly chosen fields were counted. The samples were divided into SPIN1 low-expression (< 50% positivity) and high-expression (≥50%) groups, as previously described [
11]. All slides were scanned and photographed in a Pannoramic P250 scanner (3DHistech, Hungary).
Cell culture and transfection
Human breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-468 were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (MCF-7) or Leibovitz’s L15 medium (MDA-MB-231 and MDA-MB-468), supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, U.S.). MCF-7/ADM cells were derived by treating MCF-7 cells with stepwise increasing concentrations of Adriamycin over 8 months and cultured in RPMI-1640 medium supplemented with 10% FBS. To maintain their resistance, MCF-7/ADM cells were cultured in the presence of a low concentration of Adriamycin and passaged for 1 week in drug-free medium before the experiments. The identities of the cell lines were confirmed by STR profiling in 2017 by Guangdong Hybribio Biotech Ltd. (Guangzhou, China;
http://www.hybribio.cn).
Cells were transfected with miRNA mimics (GenePharma, Shanghai, China), SPIN1 siRNA (si-SPIN1, RiboBio, Guangzhou, China) or the respective negative controls (NC), using X-tremeGENE transfection reagent (Roche, Indianapolis, IN, U.S.). The entire SPIN1 coding sequences were cloned into the expression plasmid pcDNA3.1(+) (pcDNA3.1(+)-SPIN1) and the empty plasmid pcDNA3.1(+) was used as a control. Plasmids were transfected with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.
Chemosensitivity assay
For cell chemosensitivity assay, cells (5 × 10
3) were seeded in 96-well plates. Adriamycin (Dalian Meilun, China) was added to determine the sensitivity to chemotherapy. Cell viability was detected after 48h as previously described [
11].
Microarray analysis
MCF-7/ADM cells were treated with SPIN1 siRNA or negative control for 48h, followed by RNA preparation, labeling and hybridization in NimbleGen Hybridization System. Signal intensity was calculated from digitized images captured by Axon GenePix 4000B scanner. KEGG pathway enrichment analysis was performed using the DAVID online tool. The microarray data were deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE71141).
RNA isolation and quantitative real-time PCR (qRT-PCR)
For mRNA quantitative analysis, total RNAs were prepared using Trizol (Invitrogen) and were reverse transcribed with a Rever Tra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was performed in a total volume of 10-μl SYBR Green Real-time PCR Master Mix (Roche, Mannheim, Germany) on a Bio-Rad CFX™ 96 C1000 Real-Time system. For quantitative detection of miRNA, reverse transcription and qPCR assay were performed using the All-in-One™ miRNA qRT-PCR detection kit (Genecopeia, Rockville, MD, USA). Primers for miR-148a-3p (HmiRQP0204), miR-148b-3p (HmiRQP0206), miR-152-3p (HmiRQP0213) and U6 (RNU6B, HmiRQP9001; reference gene) were from GeneCopoeia.
Luciferase assay
The pmirGLO vector (Promega) was used to construct the recombinant plasmid pmirGLO-
SPIN1 containing the
SPIN1 mRNA 3’-UTR fragments which possess binding sites of miR-148a-3p, miR-148b-3p and miR-152-3p [
11]. The luciferase assay was performed as previously described [
20].
Western blot
Briefly, whole-cell or tissue lysates were resolved by electrophoresis, and proteins were transferred to nitrocellulose membranes and blotted with antibodies against SPIN1 (1:1000, D152571, Sangon Biotech), ABCB4 (1:500, GTX47122, Genetex), CYP2C8 (1:200, sc-164136, Santa Cruz), UGT2B4 (1:2000, ab173580, Abcam), UGT2B17 (1:1000, abs110602, absin), or β-actin (1:1000, BA2305, Boster). The protein bands were detected using the chemiluminescent substrate with the AlphaView software (Version: 3.2.2.0) on a FluorChem Q machine (Cell Biosciences, Inc., Santa Clara, CA, USA).
In vivo mouse xenograft model
Four-week-old female nude mice (
n = 20) were used for xenograft studies. MCF-7/ADM cells (2 × 10
6) were transplanted into the mammary fat pads of mice. We have previously shown that miRNA-489 could directly target and suppress SPIN1 expression in breast cancer [
11]. Here we used miRNA-489 as a tool to downregulate SPIN1 expression in MCF-7/ADM cells. Lentiviruses miRNA-489 or miRNA control was stably transfected into cells. Chemosensitivity to Adriamycin was ascertained from mice injected with Adriamycin (5 mg/kg) through the tail vein weekly. Tumor growth was monitored once a week by Vernier caliper. At the 8th week, tumor masses were excised and further analyzed by immunohistochemistry, qRT-PCR and Western blot. Animal experiments were approved by the Laboratory Animal Center of Shandong University, and were conducted in accordance with the institutional guidelines.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software. The correlation between miR-148/152 family, SPIN1, and ABCB4/CYP2C8/UGT2B4/UGT2B17 expression was determined by Spearman’s correlation. Student’s t test was used to analyze the differences between two groups. The chi-square test or Fisher’s exact test was utilized to assess the relationship between SPIN1 expression and the clinical characteristics. P values < 0.05 were considered to be statistically significant.
Discussion
Here, we show that SPIN1 is upregulated in drug-resistant breast cancer cells and tissues.
SPIN1 is identified as a novel target of the miR-148/152 family and enhances Adriamycin resistance by regulating drug metabolizing enzymes and transporter CYP2C8, UGT2B4, UGT2B17 and ABCB4 in breast cancer (Fig.
5i). In addition, high expression of
SPIN1 or low expression of the miR-148/152 family predicts poorer survival in patients with breast cancer.
In this study, by ectopic expression and loss-of-function experiments of SPIN1, we indicated that SPIN1 enhanced Adriamycin resistance of breast cancer cells. We understand that in vivo experiments with a specific depletion of SPIN1 may better reveal the involvement of SPIN1 in chemoresistance, which needs more attention in our future study. Here SPIN1 was found to lead to marked upregulation of drug metabolizing enzymes and transporter. Recently, Franz et al. have found that SPIN1, in cooperation with the transcription factor MAZ, directly enhances expression of GDNF to activate the RET signaling to increase proliferation and decrease apoptosis of liposarcoma cells [
10]. These findings, together with our results, highlighted the critical roles of SPIN1 in regulating genes expression and prompted us to further investigate the underlying mechanisms of SPIN1-mediated activation of the drug metabolizing enzymes and transporter.
The drug metabolizing enzymes and transporter that identified as SPIN1 downstream effectors in this study have been shown to be highly expressed in drug-resistant tumors and associated with chemoresistance. The ATP-binding cassette (ABC) transporter gene
ABCB4 is found to be amplified in Adriamycin-resistant breast cancer cells relative to drug-sensitive cells [
34]. Sprouse et al. suggest that upregulation of CYP2C8 may account for paclitaxel resistance in the drug-resistant MDA-MB-231 breast cancer cells [
35]. The UDP-glucuronosyltransferases UGT2B4 and UGT2B17 have been linked to breast cancer risk [
36,
37]. However the upstream regulators of these drug metabolizing enzymes and transporter remain largely unknown. Here we showed that these drug metabolizing enzymes and transporter were controlled by SPIN1 and involved in breast cancer chemoresistance, which may be potential targets to reverse drug resistance in breast cancer.
We have previously found that miR-489 could directly target
SPIN1 in breast cancer [
11]. And here we further showed that
SPIN1 was also targeted by three members (miR-148a/148b/152-3p) of the miR-148/152 family [
38]. All these three miRNAs have been reported to be downregulated in breast cancer tissues and cell lines [
39‐
41]. In addition, they are all crucial modulators for many biological processes in breast cancer. MiR-148a inhibits breast cancer migration, invasion and angiogenesis by suppressing
WNT-1 [
39],
MMP-13 [
42],
ERBB3 [
43]. MiR-148b is identified as a relapse-associated miRNA and suppresses breast cancer progression by targeting a series of cancer-related oncogenes [
40]. MiR-152 inhibits tumor angiogenesis via targeting
IGF-IR and
IRS1 in breast cancer [
41]. However, the function and mechanism of the miR-148/152 family in breast cancer chemoresistance have not yet been reported. Here we found that miR-148-3p, miR-148b-3p and miR-152-3p were downregulated in Adriamycin-resistant MCF-7/ADM cells compared with the parental MCF-7 cells. The three miRNAs suppressed Adriamycin resistance of breast cancer cells by directly targeting
SPIN1. Moreover, analysis of publicly available data revealed that the miR-148/152 family was associated with patients’ survival in breast cancer. Our study demonstrated a newly-identified involvement of the miR-148/152 family in breast cancer Adriamycin resistance and further study is underway to confirm this in clinical samples.
In conclusion, we have presented evidence that SPIN1, a novel target of the miR-148/152 family, is upregulated in drug-resistant breast cancer cells and tissues and confers Adriamycin resistance by upregulating drug metabolizing enzymes and transporter in breast cancer. Our study may provide useful information for the development of alternative approaches to drug-resistant breast cancer.