Tumor Biol. DOI 10.1007/s13277-015-3375-5

RESEARCH ARTICLE

ObRb downregulation increases breast cancer cell sensitivity to tamoxifen Yingying Qian 1 & Dongmin Shi 1 & Jinrong Qiu 1 & Fang Zhu 1 & Jing Qian 1 & Shaohua He 1 & Yongqian Shu 1 & Yongmei Yin 1 & Xiaofeng Chen 1

Received: 14 January 2015 / Accepted: 24 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Leptin is a potent adipokine that plays an important role in the progression of breast cancer and interferes with the action of tamoxifen. We investigated the molecular mechanism underlying the effect of leptin on tamoxifen resistance in breast cancer cells that express leptin receptor (ObRb), and evaluated the impact of ObRb suppression on tamoxifen treatment in MCF-7 and tamoxifen-resistant (TAM-R) cells. Leptin-induced signaling pathway activation was examined by qRT-PCR and Western blotting. Chromatin immunoprecipitation assays were performed to further examine the binding of estrogen receptor (ER) α on the promoter of cyclin D1 (CCND1) gene. The effects of combined ObRb knockdown and tamoxifen treatment were evaluated in MCF-7 and TAMR cells. We found that the enhanced proliferation effects induced by leptin were related to extracellular-signal-regulated kinase (ERK) 1/2 and signal transducers and activators of transcription (STAT) 3 signaling pathway activation and CCND1 upregulation. Leptin enhanced CCND1 gene transcription by inducing the binding of ERα to the promoter of CCND1 gene. ObRb knockdown significantly enhanced the inhibitory effects of tamoxifen on TAM-R cell proliferation Yingying Qian and Dongmin Shi contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3375-5) contains supplementary material, which is available to authorized users. * Yongmei Yin [email protected] * Xiaofeng Chen [email protected] 1

Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, China

and survival. This study suggested that long-term endocrine therapy facilitates leptin and ObRb overexpression in breast cancer cells, which attenuates the inhibitory effect of tamoxifen by activating both the ERK1/2 and STAT3 signaling pathways and upregulating CCND1 gene expression. Combination therapy involving ObRb knockdown and tamoxifen treatment may be an alternative therapeutic option for tamoxifenresistant breast cancer. Keywords Leptin . ObRb . Tamoxifen resistance . Cyclin D1 . Breast cancer

Introduction Breast cancer is one of the most common malignant tumors among women all over the world [1]. About 70 % of all patients with breast cancer have estrogen receptor (ER)-positive tumors; thus, selective ER modulators such as tamoxifen are widely used for treating these patients [2, 3]. Unfortunately, up to half of all responsive tumors eventually develop resistance to tamoxifen, illustrating the fact that endocrine resistance is a crucial problem in the management of breast cancer [4–6]. Leptin, the product of the obese (OB) gene, is a cytokinelike protein primarily secreted by mature adipocytes [7, 8]. It is a multifunctional peptide hormone that can modulate wideranging biological processes, including appetite regulation, bone formation, reproductive function, immune response, and angiogenesis. Leptin exerts its function by binding to its receptor (ObR), where the long isoform ObRb contains the full-length intracellular domain and has full signaling potential [9, 10]. Recently, it was suggested that leptin might be involved in breast cancer development. Compared with noncancerous tissues, leptin and ObRb are significantly

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overexpressed in primary and metastatic breast cancer [11, 12]. In vitro studies have shown that leptin is a proliferative factor in breast cancer cells [13]. Moreover, we previously demonstrated the presence of leptin and ObRb in 79.8 and 85.1 % of breast cancer tissues, respectively. Additionally, it has been reported that leptin overexpression correlates with poor prognosis in tamoxifen-treated patients [14, 15]. However, the molecular mechanisms underlying the inhibitory effects of leptin on the action of tamoxifen in breast cancer are not clear. Ras/extracellular-signal-regulated kinase (ERK) 1/2 signaling, which orchestrates cell proliferation and survival, is one of the main signalings involved in leptin/ObRb signaling pathways [16]. Recent studies have demonstrated that the ERK1/2 signaling pathway is involved in tamoxifen resistance and that ERα acts as a key downstream effector of this pathway [17, 18]. Moreover, estrogen and leptin signaling interacts via the Janus kinase/signal transducers and activators of transcription 3 (JAK/STAT3) signaling pathway, which has been implicated in leptin-stimulated cell growth [19]. Cyclin D1 (CCND1) is a cell-cycle-regulating protein playing important roles in human cancers. Cyclin D1 gene amplification and overexpression correlate with tamoxifen resistance and poor prognosis [20, 21]. Leptin-induced increased CCND1 promoter activity is mediated through binding to activated STAT3 at the STAT binding sites [22]. Furthermore, cyclin D1 overexpression has been closely linked to ERα positivity [23]. Thus, we speculate that leptin interferes with the action of tamoxifen in breast cancer cells through activation of ERK1/2 and STAT3 signaling pathway. Leptin-mediated overexpression of cyclin D1 may also be associated with leptin-induced tamoxifen resistance in breast cancer. In the present study, we examined the molecular mechanisms underlying the contribution of leptin into tamoxifen resistance in breast carcinoma cells. Moreover, we demonstrated a therapeutic setting aimed at targeting ObRb to circumvent tamoxifen resistance in breast cancer cells.

Materials and methods Materials Leptin, tamoxifen, ERK inhibitor PD98059, STAT3 inhibitor JSI-124, E2 (as positive control) and DMSO (as negative control) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ObRb small interfering RNA (siRNA) and nontargeting control siRNA (ObRb-NC) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine 2000 was from Invitrogen Life Technologies (Carlsbad, CA, USA). EdU was from RiboBio (Guangzhou, China); CCK-8 kit was from

Keygen (Nanjing, China). Annexin V/PI apoptosis assay was from Invitrogen Life Technologies (Carlsbad, CA, USA). ChIP Assay kit was from Millipore (Temecula, CA, USA). Sources of antibodies are as follows: GAPDH ObRb from Santa Cruz Biotech, Santa Cruz, CA, USA; total ERK, STAT3, phosphorylated ERK, phosphorylated STAT3, ERα, and cyclin D1 antibody from Cell Signaling Technology, Beverly, MA, USA; and secondary antibodies conjugated with horseradish peroxidase (HRP) from Amersham Pharmacia Biotech, Piscataway, NJ, USA. Cell culture medium, DMEM, was from Biosource International, Inc. (Camarillo, CA, USA). Fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were from Gibco (NY, USA). Cell culture MCF-7 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and routinely grown in DMEM containing 10 % FBS and 1 % penicillin/streptomycin. Tamoxifen-resistant (TAM-R) cells (kindly provided by Professor Fan, University of Virginia Health Sciences System, Charlottesville, VA, USA) were maintained as described previously [24]. Transient transfections MCF-7 and TAM-R cells (1×105 cells) were seeded in sixwell plates and cultured to 70 % confluence. A 400-μL mixture of siRNA in DMEM (100 nmol/L) and Lipofectamine 2000 was incubated with the cells for 4–6 h and then replaced with culture medium containing 5 % FBS. The cells were harvested 24 or 48 h after transfection. Quantitative real-time polymerase chain reaction Total RNA was isolated from cells using TRIzol® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The ribosomal bands were visualized on a 1 % TAE agarose gel to assess RNA integrity. The purity of the RNA was validated by measuring A260/280 using BioPhotometer plus (Eppendorf, Germany). Two micrograms of total RNA was reverse transcribed into first-strand cDNA by using a reverse transcription reagent kit (Fermentas, Lithuania) according to the manufacturer’s protocol. The quantitative RTPCR was performed using the SYBR® Green real-time PCR kit (TOYOBO, Japan) according to the manufacturer’s manual on ABIStepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). All quantifications were performed with GAPDH as the internal standard. The PCR primer sequences were as follows: OBRb, forward 5′-ATAG TTCAGTCACCAAGTGC-3′ and reverse 5′-GTC CTGGAG AACTCTGATGTC-3′; cyclin D1, forward 5′-AACTACCT

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GGACCGC TTCCT-3′ and reverse 5′-CCACTTGAGCTT GTTCACCA-3′; and GAPDH, forward 5′-ACCCAGAAGA CTGTGGATGG-3′ and reverse 5′-TTCTAGACG GCAGGT CAGGT-3′. The PCR-amplified products were resolved by electrophoresis in 1.5 % agarose gels to visualize the products. Western blot Cells were lysed using lysis buffer supplemented with protease and phosphatase inhibitors, and cellular debris was removed by centrifugation at 12,000 rpm at 4 °C for 20 min. Proteins (20 μg/lane) were separated using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis following quantification with a bicinchoninic acid kit (Thermo Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes (Roche, Indianapolis, IN, USA). Blots were blocked with 5 % dry milk in Tris-buffered saline/0.1 % Tween 20 and incubated with primary antibodies: anti-ObRb (1:100), ERα (1:1000), GAPDH (1:10000), ERK1/2, P-ERK1/2, STAT3, P-STAT3, and cyclin D1 (1:1000) overnight at 4 °C and then incubated with the corresponding HRP-conjugated secondary antibodies (1:5000) for 1 h. Bands were normalized to GAPDH expression, which was used as the internal loading control. The signals were recorded using a VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA) or on photographic film. Ethynyl deoxyuridine retention assay Dissociated cells were exposed to 25 mM ethynyl deoxyuridine for 2 h at 37 °C and then fixed in 4 % paraformaldehyde. After permeabilization with 0.5 % Triton X-100, the cells were reacted with Apollo reaction cocktail for 30 min. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 30 min and visualized under fluorescence microscopy (DMI 300b; Leica, Wetzlar, Germany).

OD of individual test group / Mean OD of control group)× 100. Annexin V/propidium iodide apoptosis assay Cells were seeded in six-well plates at a density of 1 × 105 cells/well. At the end of the treatment, cells were washed with phosphate-buffered saline two times and collected by centrifuging at 500 rpm for 10 min. Cells were suspended by sequential addition of 500 μL binding buffer, 5 μL annexin V–fluorescein isothiocyanate (FITC), and 5 μL propidium iodide (PI) according to the manufacturer’s instructions. Flow cytometric data were analyzed using FlowJo 7.6 software (Miltenyi Biotec, Bergisch Gladbach, Germany) and visualized in dot plots of annexin V/FITC staining against PI staining. Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed according to the manufacturer’s protocol. Briefly, 70 % confluent MCF-7 cells were treated with 100 ng/mL leptin, 1000 nM tamoxifen, or 100 ng/mL leptin+1000 nM tamoxifen for 48 h and then fixed in 1 % formaldehyde for 15 min. Cells were lysed, and the nuclei were pelleted by centrifugation, resuspended, sonicated on ice with a sonicator to shear the cross-linked DNA to an average length of 250–750 bp, and centrifuged at 12,000 rpm to remove insoluble material. The sheared chromatin was immunoprecipitated with 2 μg anti-ERα or control immunoglobulin G antibody overnight at 4 °C. The antibodyDNA complexes were incubated with protein A/G–agarose beads, and cross-links were reversed at 65 °C in a rotating incubator for 1 h. The immunoprecipitated DNA was analyzed using PCR with CCND1-specific primers: forward, 5′-TAGGAACCTTCGGTGGTCTT-3′, and reverse, 5′-TCTAGCCTGGAGACTCTTCG-3′. Following 28–30 cycles of amplification, PCR products were separated on 2 % agarose gels, stained with ethidium bromide.

Cell cytotoxicity assay Statistical analysis Cell viability was analyzed using Cell Counting Kit-8 (CCK8). Cells (2×103) were seeded in 96-well plates and incubated for 24 or 48 h, and then 10-μL CCK-8 solution was added to each well and the cultures were incubated at 37 °C for 1 h. The absorbance of each well was read at 450 nm on an ELX-800 spectrometer reader (BioTek Instruments, Winooski, VT, USA). The percentage growth inhibition was calculated using the following formula; the concentration of tamoxifen needed to inhibit 50 % cell growth (IC50) was generated from the dose-response curves: % Growth inhibition=100−(Mean

All experiments were independently repeated three times in triplicate. Results are presented as the mean±standard error (SE) of the mean of at least three separate experiments. Comparisons between multiple groups were performed with either Student’s t test or one-way analysis of variance with a Bonferroni test. Differences were considered statistically significant if p< 0.05. All statistical analyses were performed using SPSS 18.0 software (SPSS, Chicago, IL, USA).

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Results Increased MCF-7 cell growth induced by leptin was related to ERK1/2 and STAT3 signaling pathway activation Previously, we demonstrated that leptin promoted MCF-7 cell growth by inducing increased nuclear expression of ERα, which interferes with tamoxifen inhibition of MCF-7 cells [14]. As is known, ERK1/2 and STAT3 signaling pathways are the two classic cell proliferative signaling pathways [16, 19]. To determine the potential role of the ERK and STAT3 pathways in mediating the inhibitory effects of leptin on the action of tamoxifen in MCF-7 cells, the EdU retention assay was performed to detect cell proliferation. We treated MCF-7 cells with 100 ng/mL leptin, 100 ng/mL leptin+1000 nM tamoxifen, or 1000 nM tamoxifen for 48 h and found that tamoxifen treatment alone inhibited the proliferation of MCF-7 cells significantly (p=0.028), whereas leptin attenuated this effect induced by tamoxifen (p=0.035). Moreover, the ERK inhibitor PD98059 and the STAT3 inhibitor JSI-124 abrogated the leptin-induced increased cell proliferation significantly (p=0.01; p=0.007). The potentiating effect was most obvious following combined PD98059 and JSI-124 treatment (p= 0.003; Fig. 1a and Online Resource 1). Consistent with this observation, tamoxifen treatment dramatically decreased the levels of P-ERK1/2 and P-STAT3, and this decrease was reversed by the addition of leptin which alone induced a robust phosphorylation of ERK1/2 and STAT3. In addition, simultaneous treatment with leptin and PD98059 or JSI-124 reduced the P-ERK1/2 and P-STAT3 levels (Fig. 1b). These results indicate that the promoted proliferation induced by leptin is related to ERK1/2 and STAT3 activation. Cyclin D1 overexpression was associated with intense cross-talk between leptin and tamoxifen resistance in MCF-7 cells STAT3 activated by leptin promotes cyclin D1 transcription [25]. Our data showed that leptin induced cyclin D1 messenger RNA (mRNA) and protein expression; the STAT3 inhibitor JSI-124 abolished this induction significantly, whereas the ERK inhibitor PD98059 showed less effect than JSI-124 (Fig. 2a). In addition, tamoxifen downregulated cyclin D1 mRNA and protein expression, and this downregulation could be reversed by leptin. These results suggest that cyclin D1 may be involved in leptin-mediated resistance to tamoxifen. As noted earlier, ERK signaling is generally involved in enhanced functional activation of ER, which regulates CCND1 gene expression by recruiting to the CCND1 gene promoter [26]. Given these results, it is plausible that ERα activation may be involved in leptin-induced upregulation of cyclin D1 expression. To demonstrate this, we performed

Fig. 1 Leptin-stimulated MCF-7 cell proliferation was related to ERK1/2 and STAT3 signaling pathway activation. MCF-7 cells were treated with 1000 nM tamoxifen (TAM), 100 ng/mL leptin (LEP), or 100 ng/mL LEP+1000 nM TAM. For combined treatment, cells were pretreated with 10 μM ERK inhibitor PD98059, 10 μM STAT3 inhibitor JSI-124, or PD98059+JSI-124 followed by leptin treatment. a EdU retention assay detection of cell proliferation following 48-h treatment. Values represent the means±SE of three different experiments, each performed with triplicate samples. *p