Background
Breast cancer is a leading cause of cancer death in women [
1]. Accumulating evidence has shown that obesity is a significant risk and negative prognosis factor for breast cancer [
2]. Adipose tissue plays a crucial role as an energy storage depot, and currently, there is clear evidence that adipocytes act as endocrine cells and produce various biologically active substances, such as growth factors, cytokines, adipokines and leptin [
3,
4]. In spite of the strongly suggested association between obesity and breast cancer, the underlying molecular mechanisms are not known clearly. Previous studies indicate that leptin, one of the adipokines secreted from adipocytes, is an important factor that links obesity with breast cancer [
1,
5].
Leptin, a 16-kDa single-chain proteohormone encoded by the
LEP gene, is expressed in a variety of tissues, including placenta, ovaries, mammary epithelium, bone marrow, and lymphoid tissues [
6‐
9]. Leptin acts through specific leptin receptors (ObRs) and is a key factor in controlling the biological effects of food intake, energy balance, immune, and endocrine systems, as well as ontogenesis. Most of these functions are involved in leptin-induced signals which comprise several pathways triggered by many cytokines (i.e., canonical signaling pathways: JAK2/STAT, MAPK/ERK, and PI3K/AKT kinase) [
10]. Leptin/ObRs are expressed at low levels in the epithelial cells of normal human mammary glands, but overexpressed in breast cancer cells [
9,
11,
12].
Recent studies have shown that EMT is a crucial initiator of and a contributor to tumor invasion and migration [
13]. During EMT, cancer cells undergo morphological changes, such as cell-cell junction dissolution, loss of apical-basolateral cell polarity, and acquisition of mesenchymal marker expression [
14]. Snail, Twist and ZEB, and the important transcription factors of EMT are critical points in the study the mechanism of EMT. Twist is a highly conserved transcription factor and involves in organ development, cell proliferation, differentiation and tumorigenesis [
15,
16], and it is also a major regulator in EMT and promotes tumor invasion and metastasis [
17‐
19]. Our previous study showed that leptin and interleukin 8(IL-8) induced EMT in breast cancer cells via the PI3K/AKT signal pathway [
20], and this signal pathway was a significant canonical signaling pathway in leptin-induced signals. Besides IL-8 which is involved in leptin-induced EMT, this study has found that PKM2 is another critical molecule affecting tumor progression.
Pyruvate kinase (PK) participates in the final rate-limiting step of glycolysis and catalyzes phosphoenolpyruvate(PEP) and ADP to pyruvate and ATP [
21]. PKM1, PKM2, PKL and PKR are four isoforms of PK, and they are expressed in different types of mammalian cells and tissues [
22]. PKM2 is expressed during embryonic development, but it is absent from most adult tissues [
23]. There are reports indicating that PKM2 is overexpressed in malignant cells and plays the central role not only in metabolic reprogramming but also in directed regulation of tumor progression, and PKM2 could promote EMT in colorectal cancer and hepatocellular carcinoma [
14,
24,
25].
In this study, the role of PKM2 in leptin-induced EMT in breast cancer cells is investigated; it is suggested that leptin promoted EMT in breast cancer cells via the upregulation of PKM2 expression as well as activation of PI3K/AKT signaling pathway, and PKM2 might be one of the key points and potential targets for breast cancer therapy.
Methods
Cell culture
The human breast cancer cell lines MCF-7, SK-BR-3 and MDA-MB-468 were obtained from American Type Culture Collection and maintained in DMEM supplemented with 10 % fetal bovine serum (FBS, Gibco). The cells were cultured at 37 °C in a humidified incubator with 5 % CO2.
Immunofluorescence analysis
MCF-7, SK-BR-3 and MDA-MB-468 cells were grown on coverslips. Cells were washed with PBS, fixed with 4 % paraformaldehyde at room temperature for 20 min, permeabilized with 0.3 % Triton X-100, and blocked with 5 % goat serum for 30 min. All cells were incubated overnight at 4 °C with the corresponding primary antibodies(OBR mouse anti-human) in blocking solution, washed three times with PBS, and incubated for 1 h in darkness at room temperature with secondary antibodies (TRITC-conjugated goat anti-mouse). After washing, nuclei were stained with DAPI for 10 min in darkness at room temperature, and then washed three times with PBS and the coverslips were mounted with 95 % glycerol. Cell fluorescence was examined using a fluorescence microscopy (Olympus, Japan).
RNA isolation and quantitative real-time (qRT)-PCR Assay
Total RNA was isolated using Trizol reagent (TAKARA, Japan) according to the manufacturer’s protocol. RNA was stored at −80 °C after being eluted with RNase-free water. RNA concentrations were measured by spectrophotometer and RNA was reversely transcribed to cDNA using reverse transcription kit (TAKARA, Japan). Real-time (RT)-PCR was implemented using iTaq Universal SYBR Green One-step kit (BioRad). Results were normalized to the endogenous β-actin mRNA. The following primers were used: PKM2 sense 5’-CCATCCTCTACCGGCCCGTTG’, PKM2 antisense 5’- CCAGCCACAGGATGTTCTCGTC-3’, β-actin sense 5’-CCTTCCTGGGCATGGAGTCCT-3’, β-actin sense 5’-CCTTCCTGGGCATGGAGTCCT-3’, β-actin antisense 5’- GGAGCAATGATCTTGATCTTC-3.
Cell transfection and infection
MCF-7 and SK-BR-3 were placed in 6-well plates at 2 × 105 per well. Twenty-four hours after placing, siRNA against PKM2 and negative miRNA (GenePharma, Shanghai) were transfected to the cells using lipofectamine 2000 liposomes (Invitrogen) according to the manufacturer’s protocol. After culturing for 48 h, transfection efficiency was quantified by Western blotting. The sequences of siRNAs were as follows: PKM2 sense 5’- CAUCUACCACUUGCAAUUATT-3’, anti-sense 5’- UAAUUGCAAGUGGUAGAUGTT-3’; negative control sense 5’-UUCUCCGAACGUGUACGUTT’, anti-sense 5’- ACGUGACACGUUCGGAGAATT-3’. Lentivirus-based short hairpin RNA (shRNA) vector and lentivirus-based cDNA targeting the PKM2 gene were constructed by GenePharma (Shanghai, China). PKM2 shRNA was generated with CATCTACCACTTGCAATTA oligonucleotide targeting exon 10 of the PKM2 transcript. Lentiviru vector shRNA was generated with CATCTACCACTTGCAATTA oligonucleotide. Cells were selected with puromycin for 14 days at 37 °C after being infected with lentiviral. The effectiveness of transfection using lentiviruses in SK-BR-3 cells was verified by Western blotting.
Western blot
All proteins from cancer cells were subjected to SDS-PAGE and then transferred to PVDF membrane. Blots were probed with PKM2, p-PKM2, p-AKT (Ser473), AKT antibody (Cell Signaling Technologies, USA), OBR antibody (Santa, USA), E-cadherin, vimentin, and fibronectin antibody (Bioword, USA). Membranes were analyzed using Enhanced Chemiluminescence (ECL) detection system (VIAGENE, USA).
Wound healing migration and matrigel invasion assays
For wound healing migration assays, cells were seeded onto 6-well plates. After treatment, the cell monolayer was scratched with a pipette tip and then washed three times to remove the floating cells. Then, fresh serum-free medium with leptin was added, and photos were taken at 0, 24, 36 and 72 h using a microscope (Olympus, Japan). Scratch areas were measured using image software. For invasion assays, tumor cells were performed using transwell system (Millipore, USA) with 8 μm-pore polycarbonate filter membrane. The chambers were pre-coated with 50 μl matrigel (1:7 dilution; Sigma, USA). The upper chambers were seeded with 2 × 104 tumor cells in serum-free medium and the lower chambers were filled with medium containing 15 % fetal bovine serum as a chemo-attractant. After being incubated for 24 h, cells on the interior of upper chamber were scrubbed and the invading cells in the lower chambers were fixed with 4 % paraformaldehyde. Then, the polycarbonate membranes were stained with 0.1 % crystal violet for 10 min at room temperature. The number of invasion cells in five randomly selected fields under microscope (Olympus, Japan) was counted.
Xenografts of nude mice
Breast cancer cells (5 × 106/per inoculation) were injected into mammary fat pads of 5-week-old female nude mice (n = 9/group). Tumor volumes were measured using calipers, and defined as ab2/2(a: length, b: width). Mice were sacrificed when 5 weeks of post-injection, and tumors and lungs were removed, fixed in 4 % formaldehyde, and emdedded in paraffin. Tumor formation and proteins were detected by H&E staining and immunohistochemical staining. This study was approved by the Ethical Committee of Chongqing Medical University.
Immunohistochemistry
The expression of E-cadherin, vimentin, fibronectin, Twist and PKM2 in xenografts of nude mice were determined using immunohistochemistry. After being deparaffinized, tissue paraffin sections were heat-treated with citrate buffer (0.01 mol/pH6.0) as an epitope retrieval protocol. Endogenous peroxidase was blocked with 3 % H2O2 for 15 min, followed by rinsing twice, and then sections were incubated with 5 % goat serum for 30 min to avoid non-specific binding. After that, sections were incubated with PKM2 antibody (Cell Signaling Technologies, USA), E-cadherin, vimentin and fibronectin antibody (Bioword, USA) (1:200 dilution; 5 % BSA) at 4 °C overnight. Subsequently, sections were washed with PBS 5 times, and incubated with HRP-conjugated secondary antibody (BiYunTian, China) for 1 h. The color was developed using 3-3’-diaminobenzidine after being washed 5 times with PBS. Counterstaining was performed with hematoxylin. Finally, sections were dehydrated and mounted with a neutral resin.
Statistical analysis
All experiments were replicated thrice and data were expressed as mean ± standard deviation. All statistical analyses were performed with SPSS 19.0 software. The statistical significance between each group was analyzed using Student’s t-test or one-way ANOVA. P < 0.05 was considered as statistically significant.
Discussion
Epithelial-mesenchymal transition (EMT) has been implicated in tumor cells invasion, metastasis, apoptosis, stemness and treatment failure [
26,
27]. Leptin, an established risk factor for many cancers, has been found to act in the cell cycle, proliferation, tumor development, and progression [
28‐
30]. Our previous studies demonstrated that leptin could promote EMT in breast cancer cells [
20], and IL-8 was one of the key molecules which affected leptin-mediated EMT. To explore other key regulatory molecules that are involved in leptin-induced EMT of breast cancer cells, gene expression chip array was conducted, which found that a series of enzymes related to glycometabolism increased in breast cancer cells, such as PGK1, PGAM2, PDK2, SUCLG1, DLST, and PKM2. As one of the most increased molecules, PKM2 was verified to increase significantly in MCF-7 and SK-BR-3 cells treated with leptin. Thereby, we hypothesized that leptin promoted epithelial-mesenchymal transition of breast cancer cells via the upregulation of pyruvate kinase M2.
Pyruvate kinase M2 (PKM2), which is a key enzyme of glycometabolism and can act as a transcriptional co-activator, is overexpressed in multiple cancer types and involved in the Warburg effect [
31‐
33]. Studies have shown that PKM2 controls chromosome segregation, cell-cycle progression, cells proliferation, EMT, and tumorigenesis [
34‐
36]. It is firstly revealed in this study that leptin not only induces breast cancer cells EMT, but also upregulates PKM2 expression; however, the underlying mechanism remains unknown.
To this end, firstly, canonical signaling pathways inhibitors, PD98059, AG490 and LY294002, were adopted to explore the signaling pathways involved in leptin-induced breast cancer cells EMT. It was found that LY294002 blocked leptin-induced PKM2, p-PKM2, p-AKT as well as EMT-associated marker expression in MCF-7 and SK-BR-3 cells. As reported in other studies, our results also indicated that PI3K/AKT signaling pathway was involved in EMT [
37,
38]. In addition, siPKM2 abolished EMT-associated marker expression and inhibited leptin-induced migration and invasion of those breast cancer cells. So it strongly suggested that leptin could upregulate PKM2 in MCF-7 and SK-BR-3 cells, and PKM2 played a significant role in EMT induction. It is well known that PKM2 also functions as a transcriptional coactivator, and the regulatory mechanism between PI3K/AKT pathway and PKM2 molecular should be elucidated in further researches.
It has been reported that Twist is the key transcriptional factor by down-regulating E-cadherin to promote EMT, cell motility and invasiveness [
17,
39]. In addition, increased Twist expression is found in multiple tumors including melanoma [
40], prostate [
39], pediatric osteosarcoma [
41], gastric [
42] and breast cancer [
17,
43]. The overexpression of Twist positively correlates with tumor aggressiveness and poor prognosis [
17,
39,
40]. Therefore, Twist was detected during EMT of MCF-7 and SK-BR-3 cells induced by leptin, and our data showed that leptin upregulated Twist expression, but this effect was weakened by PKM2-siRNA. The potential mechanism in the interaction of PKM2 with Twist needs to be studied.
Xenograft-bearing mouse models showed that RNA interference against PKM2 obviously decreased tumor volume and weight, increased the survival rate of mice, and weakened liver and lung metastases. Besides, the decreased expression of EMT associated markers and Twist were found by IHC staining of tumor tissue.
Conclusions
This study found that leptin didn’t only induce breast cancer cells EMT, but also upregulated PKM2 expression, which resulted from the activation of PI3K/AKT pathway. Our findings also indicated the crucial role of PKM2 in leptin-mediated EMT, and PKM2 might be one of the key points and potential targets for breast cancer therapy.
Acknowledgments
We thank Zhimin Lu (The University of Texas MD Anderson Cancer Center) for his insightful suggestions.