BMB Reports 2019; 52(6): 385-390  https://doi.org/10.5483/BMBRep.2019.52.6.189
Leptin stimulates IGF-1 transcription by activating AP-1 in human breast cancer cells
Dong Yeong Min1, Euitaek Jung1, Juhwan Kim1, Young Han Lee1,2, and Soon Young Shin1,2,*
1Department of Biological Sciences, Sanghuh College of Lifesciences, Koknkuk University, Seoul 05029, Korea, 2Cancer and Metabolism Institute, Konkuk University, Seoul 05029, Korea
Correspondence to: *Tel: +82-2-2030-7946; Fax: +82-2-3437-9781; E-mail: shinsy@konkuk.ac.kr
Received: August 17, 2018; Revised: September 11, 2018; Accepted: October 4, 2018; Published online: June 30, 2019.
© Korean Society for Biochemistry and Molecular Biology. All rights reserved.

cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Leptin, an adipokine regulating energy metabolism, appears to be associated with breast cancer progression. Insulin-like growth factor-1 (IGF-1) mediates the pathogenesis of breast cancer. The regulation of IGF-1 expression by leptin in breast cancer cells is unclear. Here, we found that leptin upregulates IGF-1 expression at the transcriptional level in breast cancer cells. Activating protein-1 (AP-1)-binding element within the proximal region of IGF-1 was necessary for leptin-induced IGF-1 promoter activation. Forced expression of AP-1 components, c-FOS or c-JUN, enhanced leptin-induced IGF-1 expression, while knockdown of c-FOS or c-JUN abrogated leptin responsiveness. All three MAPKs (ERK1/2, JNK1/2, and p38 MAPK) mediated leptin-induced IGF-1 expression. These results suggest that leptin contributes to breast cancer progression through the transcriptional upregulation of leptin via the MAPK pathway.

Keywords: Activating protein-1, Breast cancer, Insulin-like growth factor-1, Leptin, Mitogen-activated protein kinase
INTRODUCTION

Leptin is a peptide hormone encoded by the leptin (LEP) gene, a homolog of the mouse obese (ob) gene (1). It is mainly produced by adipocytes and mainly affects the hypothalamus to decrease food intake and modulate glucose and fat metabolism (2, 3). Mice with a homozygous mutation in ob (ob/ob) become massively obese (3). Leptin is also produced by various peripheral tissues, including placenta, ovaries, skeletal muscle, and mammary epithelial cells (4). In addition to energy metabolism, leptin also has numerous biological activities, including in immune function, cell proliferation, angiogenesis, metastasis, and apoptosis (2). In breast cancer cells, leptin stimulates proliferation (58). Additionally, overexpression of leptin and leptin receptor (Ob-R) is correlated with a high risk of tumor recurrence (9) and closely associated with the poor prognosis of breast cancer (10, 11). These studies suggest that the leptin signal contributes to promoting breast cancer progression (12).

Ob-R belongs to the type I cytokine receptor family and transmits signals through the JAK-STAT pathway (13). Leptin-regulated genes have been identified using microarray systems in MCF-7 breast cancer cells (14). They found that cell cycle regulators (e.g., cyclin D1) and anti-apoptotic genes (e.g., BCL2 and survivin) are involved in leptin-induced cell proliferation. Additionally, leptin may induce growth factor secretion. A possible candidate causing these effects is insulin-like growth factor-1 (IGF-1). IGF-1 is a potent mitogen in nearly every cell in the body (15). Numerous studies have demonstrated that IGF-1 stimulates cell proliferation, migration, and metastasis and has critical roles in the development of human breast cancer (16, 17). High levels of circulating IGF-1 are closely associated with high risks of prostate and colorectal cancer (18, 19). A recent meta-analysis revealed a positive association between high concentrations of IGF-1 and increased breast cancer risk (20), indicating that IGF-1 is an important growth factor in the development of breast cancer.

Several studies have shown the effect of leptin on IGF-1 expression in hepatocytes (21, 22) and that a positive relationship between leptin and IGF-1 expression; for example, IGF-I can induce the transcriptional activation of the leptin gene in MCF7 cells (11). However, the mechanism underlying leptin regulation of IGF-1 gene expression is unclear in breast cancer cells.

This study aimed to investigate how leptin affects IGF-1 expression in breast cancer cells. We found that leptin stimulates IGF-1 gene expression by activating AP-1 cis-acting elements spanning nucleotides −39 to −27 upstream of the transcription initiation site of IGF-1 in human breast cancer cells.

RESULTS AND DISCUSSION

Upregulation of IGF-1 transcription by leptin in breast cancer cells

The IGF-1 expression is stimulated by various signals, including growth hormone (23, 24), thyroid hormone (24), epidermal growth factor (25), and parathyroid hormone (26). However, it is unclear whether leptin stimulates IGF-1. To investigate whether leptin regulates IGF-1 expression in breast cancer cells, two breast cancer cell line, estrogen receptor (ER)-positive MCF-7 cells and ER-negative MDA-MB-231 cells, were treated with leptin for various times and IGF-1 mRNA expression was measured. Real-time PCR analysis showed that IGF-1 mRNA expression peaked at 6 h and then slowly decreased after leptin stimulation in MCF-7 cells (Fig. 1A). Similar results were obtained in MDA-MB-231 cells (Fig. 1B). Leptin-induced elevation of IGF-1 protein levels was confirmed by immunoblotting in MCF-7 cells (Fig. 1C) and MDA-MB-231 cells (Fig. 1D). Upon leptin stimulation, increased IGF-1 protein levels were easily detectable at 24 h, after which IGF-1 gradually accumulated until 48 h. Immunofluorescence microscopy also revealed strong intensities of IGF-1 staining compared to the vehicle-treated control in MCF-7 cells (Fig. 1E). These data demonstrate that leptin upregulates IGF-1 expression in both ER-negative and ER-positive breast cancer cells.

Leptin stimulates IGF-1 promoter activity through AP-1 cis-acting elements within the 5’-regulatory region of IGF-1

The 5’-regulatory region of human IGF-1 contains several putative cis-acting regulatory elements (27). To determine whether leptin upregulates IGF-1 expression at the transcriptional level and to identify the leptin-responsive cis-acting element in the IGF-1 promoter, we generated a series of deletion constructs of the 5’-flanking region of human IGF-1 linked to the luciferase reporter gene and transfected them into MCF-7 cells. As shown in Fig. 2A, leptin triggered a significant increase in IGF-1 promoter reporter activity. The promoter-reporter harboring the sequence from nucleotides −95 to −3 was still capable of inducing reporter activity. These data suggest that leptin response elements are located within this region.

To identify the cis-acting element responsible for leptin-induced stimulation of the IGF-1 promoter, we analyzed genomic sequences between nucleotide −95 and −3 using the web-based transcription factor search tool MatInspector (http://www.genomatix.de/). The result shows that a putative AP-1-binding motif (5’-TCCTTACTCAATA-3’) spanning from nucleotide −39 to −27 (Fig. 2A). AP-1 is a well-known transcription factor complex consisting of homo- or heterodimers of Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) (28). AP-1 contributes to the proliferation and transformation of breast cells (29) and is involved in leptin-induced aromatase expression, which catalyzes estrogen biosynthesis, in MCF-7 cells (30). However, the effects of AP-1 on IGF-1 expression have not been characterized.

To evaluate the role of this putative AP-1-binding site in leptin-induced IGF-1 expression, we introduced a site-directed mutation in the AP-1-binding sequence using the pIGF1-Luc (−95/−3) construct. Disruption of the AP-1-binding core sequence (mtAP1; ACTC to ACTG) resulted in a significant loss of leptin-stimulated promoter activity compared to the wild-type (WT) construct (Fig. 2B). These data suggest that the putative AP-1-binding site at −39/−27 is involved in leptin-induced IGF-1 promoter activation.

To assess the role of AP-1 in regulating IGF-1 promoter activity, we transfected the −95/−3 construct into MCF-7 cells, along with an expression plasmid for AP-1 components. Forced expression of c-Fos (Fig. 2C) or c-Jun (Fig. 2D) stimulated promoter reporter activity in a plasmid concentration-dependent manner. These results suggest that AP-1 can trans-activate the IGF-1 promoter in MCF-7 cells.

Knockdown of c-Fos or c-Jun by shRNA abrogates leptin-induced IGF-1 mRNA expression

To further verify the role of AP-1 in leptin-induced IGF-1 promoter activity, we established MCF-7 variant cell lines expressing lentiviral shRNA against scrambled control (MCF7/shCT), c-Fos (MCF7/shFos), and c-Jun (MCF7/shJun). Stable knockdown of basal and leptin-induced expression of c-Fos (Fig. 3A) or c-Jun (Fig. 3B) was confirmed after leptin treatment in serum-starved cells. The ability of leptin to induce accumulation of IGF-1 protein was substantially attenuated when either c-Fos or c-Jun expression was reduced compared to in MCF7/shCT cells (Fig. 3C). Real-time PCR analysis shows that leptin elevated IGF-1 mRNA expression by 6.33 ± 1.04-fold in MCF7/shCT cells, which was reduced to 2.4 ± 0.6- and 1.7 ± 0.43-fold in MCF7/shFos and MCF7/shJun cells, respectively (Fig. 3D). These results demonstrate that AP-1 contributes to leptin-induced IGF-1 mRNA expression.

Leptin induces AP-1 expression through multiple MAP kinase pathways

It has been demonstrated that leptin induces AP-1 expression by activating the extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK) pathway to induce aromatase mRNA expression in MCF-7 cells (30). We confirmed that leptin increased the levels of both c-Fos and c-Jun proteins within 30 min after leptin stimulation in MCF-7 cells (Fig. 4A). To delineate the signal pathway mediating leptin-induced AP-1 expression, we treated serum-starved MCF-7 cells with leptin at various times, and the phosphorylation status of MAPKs was assessed using phospho-specific antibodies. The increases in phosphorylated levels of ERK1/2 at Thr202/Tyr204 and p38 MAPK at Thr180/Tyr182 were maximal within 30 min, while JNK1 at Thr183/Tyr185 was evident within 30 min and then progresively increased to 120 min after leptin treatment (Fig. 4B). To determine which MAPK signaling pathway is necessary for leptin-induced AP-1 expression, we used chemical inhibitors against MAPKs. Pretreatment of MCF-7 cells with the MAPK kinase (MEK) inhibitor U0126 led to a substantial reduction in leptin-induced accumulation of both c-Fos and c-Jun proteins (Fig. 4C). In contrast, pre-treatment with the p38 MAPK inhibitor SB203580 had a minimal effect, while the JNK inhibitor SP600125 increased c-Fos but decreased c-Jun protein levels, suggesting that multiple MAPKs differentially regulate AP-1 expression. We next examined whether inhibition of each MAPK affects leptin-induced IGF-1 protein accumulation. Treatment with all three MAPK inhibitors substantially decreased the accumulation of IGF-1 protein (Fig. 4D). To precisely quantify the effect of MAPK inhibitors, we performed real-time PCR analysis. Upon leptin stimulation, the IGF-1 mRNA level was enhanced by 6.8 ± 0.7-fold, which was significantly reduced to 1.1 ± 0.31-, 2.7 ± 0.27-, and 3.6 ± 0.2–46-fold (n = 3, all P < 0.0001) in the presence of U0126, SB203580, and SP600125, respectively (Fig. 4E). These data suggest that multiple MAPK pathways are important in leptin-induced IGF-1 expression dependently or independently of AP-1.

The present study shows that leptin stimulates AP-1-mediated IGF-1 expression in breast cancer cells. We found that (i) disruption of the AP-1-binding site spanning from nucleotides −39 to −27 within the IGF-1 promoter region attenuated leptin-induced IGF-1 promoter activity, (ii) exogenous expression of either c-Fos or c-Jun stimulated the IGF-1 promoter, and (iii) knockdown of either c-Fos or c-Jun abrogated leptin-induced IGF-1 expression. Additionally, we found that all three major MAPKs, ERK1/2, JNK1/2, and p38 MAPK, which differentially regulate AP-1 expression, are necessary for leptin-induced IGF-1 expression in MCF-7 breast cancer cells. The previous study has demonstrated that combined treatment with leptin and IGF-1 increases proliferation as well as migration and invasion of MCF-7 and MDA-MB-231 breast cancer cells (31). We also observed that the combination of leptin and IGF-1 led to a synergistic increase in cell proliferation when compared with either treatment alone (Supplemental Fig. S1). As downstream signaling of leptin and IGF-1 involves PI3K-AKT and MAPK signalings, leptin might share a common mechanism of action with IGF-1 (32, 33). Moreover, leptin and IGF-1 synergistically transactivate epidermal growth factor receptor (EGFR) in breast cancer cells (31). Thus, it seems likely that leptin and leptin-induced IGF-1 are thought to promote the progression of breast cancer synergistically. Our results further support the role of leptin in crostalk with IGF-1 to contribute to the progression of breast cancer.

MATERIALS AND METHODS

Cells and cell culture

MCF-7 and MDA-MB-231 human breast cancer cells were from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Reagents

Human leptin was obtained from Peprotech (Rocky Hill, NJ, USA). U0126, SB203580, and SP600125 were purchased from Calbiochem (San Diego, CA, USA). The firefly and Renilla Dual-Glo™ Luciferase Assay System was purchased from Promega (Madison, WI, USA). The pRL-null plasmid, which encodes Renilla luciferase, was purchased from Promega. Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Reverse transcription-PCR and quantitative real-time PCR

Total RNA was extracted using a TRIzol RNA extraction kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real time-PCR was performed on an iCycler iQ™ system (Bio-Rad) according to the manufacturer’s recommendations. The reaction mixture (20 μl) contained 10 μl of iQ™ SYBR® Green Supermix (Bio-Rad). Gene-specific IGF-1 PCR primers were as follows: forward, 5’ GTCTGATAA TCTTGTTAGTCTATA-3’; reverse, 5’-CACAGATGGAATCTTGTG-3’. GAPDH PCR primers were: forward, 5’-CCAAGGAGTAAGAAACCCTGGAC-3’; reverse, 5’-GGGCCGAGTTGGGATAGGG-3’. The specificity of real-time PCR was verified by melting curve analysis. GAPDH was used to normalize the RNA levels in the tested samples.

Immunoblot analysis

Antibodies against GAPDH, c-Fos, and c-Jun were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), and phospho-JNK1/2 (Thr183/Tyr185) were obtained from Cell Signaling Technology (Danvers, MA, USA). IGF-1 antibody was obtained from Gene Tex (Irvine, CA, USA). Cells were lysed and separated as described previously (34). The blots were incubated with the primary and secondary antibodies and developed using an enhanced chemiluminescence detection system. Relative band intensity was quantified using ImageJ version 1.52a software (National Institute of Health, Bethesda, MD, USA) and was expressed as a ratio to GAPDH.

Immunofluorescence microscopy

MCF-7 cells cultured on coverslips were either treated with phosphate-buffered saline (PBS) or 100 ng/ml leptin for 30 min, followed by fixation, permeabilization, and incubation of primary antibodies specific to αβ-tubulin and IGF-1. After 2 h, the cells were incubated with Alexa Fluor 488-conjugated (green signal) or Alexa Fluor 555-conjugated (red signal) secondary antibody for 30 min as described previously (35). Nuclear DNA was stained with 1 μg/ml Hoechst 33258 for 10 min (blue signal). Fluorescent staining of cells was examined under an EVOS FL fluorescence microscope (Advanced Microscopy Group; Bothell, WA, USA).

Generation and mutagenesis of human IGF-1 promoter-reporter constructs

An IGF-1 promoter fragment spanning nucleotides −906 to −3 upstream of the transcription start site was synthesized from human genomic DNA (Promega) by PCR using the primers 5’-CAA AACAGC TGGCTTGGA CC-3’ (forward primer; −906F) and 5’-TCTCTCTCCCTCTTCTGGCA-3’ (reverse primer; −3R). The amplified PCR products were ligated into a T&A vector (RBC Bioscience, Taipei County, Taiwan) and then digested with KpnI and BglII. The products were ligated into the KpnI and BglII sites of the pGL4-basic vector (Promega), yielding pIGF1-Luc (−906/−3). A series of deletion constructs of human IGF-1 promoter fragments was synthesized by PCR using the pIGF1-Luc (−906/−3) construct as a template plasmid. Forward primer sequences were 5’-CCTCATCGCAGAGAAAAAG-3’ (−536 to −3), 5’-CCCCAGTCACTTCAGGGTTA-3’ (−384 to −3), and 5’-TGCTCTAGTTTTAAAATGCAAAGG-3’ (−95 to −3). One reverse primer, −3R, was used to generate all deletion constructs. The PCR products were ligated into the T&A vector, followed by digestion with KpnI and BglII. The products were ligated into the same sites of the pGL4-basic vector. Site-specific mutation of the AP-1-binding site (mtAP1) was performed using the QuickChange site-directed mutagenesis system (Stratagene, La Jolla, CA, USA) with the −95/−3 construct used as a template plasmid. Primer sequences used to generate the point mutation were as follows: mtAP1 forward, 5’-AATAACTTTGCCAGAAGAGGGAGAGA-3’; mtAP1 reverse 5’-CAGTAAGGACTTTTTTGGGCATGGTG-3’. PCR conditions were as follows: hold for 4 min at 95°C, followed by 30 cycles consisting of denaturation at 95°C (30 s), annealing at 55°C (1 min), and elongation at 60.3°C (3 min). The point mutation site was verified by DNA sequencing (Macrogen, Seoul, Republic of Korea).

IGF-1 promoter reporter assay

MCF-7 cells were seeded into 12-well plates and transfected with 0.1 μg of the IGF-1 promoter construct using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. Where indicated, co-transfected concentrations of mammalian expression vectors for c-Fos (pCDNA3.1/Fos) or c-Jun (pCDNA3.1/Jun) were included. At 24 h post-transfection, firefly luciferase activity was measured using the Dual-Glo™ Luciferase Assay System (Promega). The relative level of luciferase activity in the untreated cells was designated as 1. Luminescence was measured with a dual luminometer (Centro LB960; Berthold Tech, Bad Wildbad, Germany).

Stable knockdown of c-Fos and c-Jun by using shRNA

MISSION shRNA Lentiviral Transduction Particles targeting c-Fos (SHCLNV-NM_005252) or c-Jun (SHCLNV-NM_002228) were introduced into MCF-7 cells according to the manufacturer’s instruction (Sigma-Aldrich). After 2 weeks, silencing of STAT3, c-Fos or c-Jun expression was determined by immunoblotting.

Statistical analysis

Statistical significance was analyzed by one-way analysis of variance followed by Sidak’s multiple comparisons test or by Dunnett’s multiple comparisons test using GraphPad Prism version 7.04 software (GraphPad Software, Inc., La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant.

SUPPLEMENTARY INFORMATION
ACKNOWLEDGEMENTS

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant no. 2016R1A2B4008570), Republic of Korea. This paper was supported by the KU Research Professor Program of Konkuk University and by the Konkuk University Researcher Fund in 2018.

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures
Fig. 1. Effect of leptin on IGF-1 expression in breast cancer cells. (A, B) MCF-7 (A) and MDA-MB-231 (B) cells were treated with 100 ng/ml leptin for various times (0–12 h). Total RNA was isolated, and IGF-1 mRNA was measured by real-time PCR. GAPDH mRNA level was used as an internal control. Bars represent mean ± S.D. (n = 3). **P < 0.001; by Sidak’s multiple comparison test. (C, D) MCF-7 (C) and MDA-MB-231 (D) cells were treated with 100 ng/ml leptin for various times (0–48 h). Whole cell lysates were measured by immunoblotting using an antibody against IGF-1. GAPDH level was examined as an internal control. The band intensities of IGF-1 relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0069; **P = 0.0006; ***P < 0.0001; #P = 0.0098; NS, not significant; by Dunnett’s multiple comparisons test. (E) MCF-7 cells were treated with PBS or 100 ng/ml leptin, and then incubated with antibodies against α/β-tubulin and IGF-1 for 2 h, followed by incubation with Alexa Fluor 488-conjugated (green signal for α/β-tubulin) or Alexa Fluor 555-conjugated (red signal for IGF-1) secondary antibody for 30 min. Nuclear DNA was stained with 1 μg/ml Hoechst 33258 for 10 min (blue signal). Bar indicates 50 μm.
Fig. 2. Role of AP-1-binding element in leptin-induced IGF-1 promoter activation. (A) MCF-7 cells were transfected with 0.2 μg of a series of 5’-deletion constructs of IGF-1 promoter reporter plasmids. Putative AP-1-binding sequence is located at −39 to −27 nt. Bars represent mean ± S.D. (n = 3). ***P < 0.0001; by Sidak’s multiple comparison test. (B) MCF-7 cells were transfected with 0.2 μg of wild-type (WT) pIGF1-Luc (−95/−3) or AP-1 site mutant construct (mtAP1). After 48 h, the cells were treated with either PBS or 100 ng/ml leptin for an additional 8 h, and luciferase activities were measured. Bars represent the mean ± SD (n = 3). ***P < 0.0001; NS, not significant; by Sidak’s multiple comparison test. (C, D) MCF-7 cells were co-transfected with pIGF1-Luc (−95/−3) construct and increasing concentrations of expression plasmid (0–200 ng) for c-FOS (C) or c-JUN (D). After 48 h, the cells were collected, and luciferase activities were measured. Bars represent the mean ± SD (n = 3). Exogenous expression of c-FOS (C) or c-JUN (D) was confirmed by immunoblotting (bottom panels).
Fig. 3. Effect of c-Fos or c-Jun knockdown on leptin-induced IGF-1 expression. (A, B) MCF-7 variant cells expressing scrambled (shCT), c-Fos shRNA (shFos), or c-Jun shRNA (shJun) were treated with 100 ng/ml leptin for 1 h. Cell lysates were immunoblotted with an antibody against c-FOS (A) or c-JUN (B). GAPDH level was examined as an internal control. The band intensities of c-FOS or c-JUN relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.001; **P = 0.0003; ***P = 0.0158; #P = 0.0079; NS, not significant; by Sidak’s multiple comparison test. (C) MCF7/shCT, MCF7shFos, and MCF7/shJun cells were treated with 100 ng/ml leptin for 12 h. Whole cell lysates were measured by immunoblotting using an antibody against IGF-1. GAPDH level was examined as an internal control. The band intensities of IGF-1 relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0043; **P = 0.0060; ***P < 0.0001; NS, not significant; by Sidak’s multiple comparison test. (D) MCF7/shCT, MCF7shFos, and MCF7/shJun cells were treated with 100 ng/ml leptin for 6 h. Total RNA was isolated, and IGF-1 mRNA was measured by real-time PCR. GAPDH mRNA level was examined as an internal control. Bars represent mean ± S.D. (n = 3). *P = 0.0318; ***P < 0.0001; NS, not significant; by Sidak’s multiple comparison test.
Fig. 4. Differential role of MAPK pathways in leptin-induced AP-1 expression. (A) MCF-7 cells were treated with 100 ng/ml leptin for various times (0–120 min). Cell lysates were immunoblotted with an antibody against c-Fos or c-Jun. GAPDH level was examined as an internal control. The band intensities of c-FOS or c-JUN relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0016; ***P < 0.0001; NS, not significant; compared with control (0 min) by Dunnett’s multiple comparisons test. (B) MCF-7 cells were treated with 100 ng/ml leptin for various times (0–120 min). Cell lysates were immunoblotted using a phospho-specific antibody against ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182), or JNK1/2 (Thr183/Tyr185) MAPK. GAPDH level was examined as an internal control. The band intensities of phosphorylated MAPKs relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0095; **P = 0.0026; ***P < 0.0001; NS, not significant; compared with control (0 min) by Sidak’s multiple comparison test. (C) MCF-7 cells were pre-treated with U0126 (10 μM), SB203580 (20 μM), or SP600125 (20 μM) for 30 min before stimulation with 100 ng/ml of leptin. After 1 h, cell lysates were prepared and immunoblotted using an antibody against c-Fos or c-Jun. GAPDH level was examined as an internal control. The band intensities of c-FOS or c-JUN relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0123; **P = 0.0038; ***P = 0.0430; #P = 0.0005; ##P < 0.0001; NS, not significant; by Sidak’s multiple comparison test. (D) MCF-7 cells were pre-treated with U0126 (10 μM), SB203580 (20 μM), or SP600125 (20 μM) for 30 min before stimulation with 100 ng/ml of leptin. After 12 h, whole lysates were prepared, and IGF-1 protein level was measured by immunoblot analysis. The band intensities of IGF-1 relative to GAPDH were measured using ImageJ software. Bars represent mean ± S.D. (n = 3). *P = 0.0074; **0.0034; ***P < 0.0001; #P = 0.0002; NS, not significant; by Sidak’s multiple comparison test. (E) Cells were treated as in (D). Total RNA was isolated and real-time PCR analysis was carried out. GAPDH mRNA level was examined as an internal control. Bars represent mean ± S.D. (n = 3). *** P < 0.0001 by Sidak’s multiple comparison test.
References
  1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425-432.
    Pubmed CrossRef
  2. Huang L, and Li C (2000) Leptin: a multifunctional hormone. Cell Res 10, 81-92.
    Pubmed CrossRef
  3. Friedman JM, and Halaas JL (1998) Leptin and the regulation of body weight in mammals. Nature 395, 763-770.
    Pubmed CrossRef
  4. Margetic S, Gazzola C, Pegg GG, and Hill RA (2002) Leptin: a review of its peripheral actions and interactions. Int J Obes Relat Metab Disord 26, 1407-1433.
    Pubmed CrossRef
  5. Hu X, Juneja SC, Maihle NJ, and Cleary MP (2002) Leptin--a growth factor in normal and malignant breast cells and for normal mammary gland development. J Natl Cancer Inst 94, 1704-1711.
    Pubmed CrossRef
  6. Laud K, Gourdou I, Pessemesse L, Peyrat JP, and Djiane J (2002) Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line. Mol Cell Endocrinol 188, 219-226.
    Pubmed CrossRef
  7. Okumura M, Yamamoto M, and Sakuma H (2002) Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta 1592, 107-116.
    Pubmed CrossRef
  8. Somasundar P, Yu AK, Vona-Davis L, and McFadden DW (2003) Differential effects of leptin on cancer in vitro. J Surg Res 113, 50-55.
    Pubmed CrossRef
  9. Ishikawa M, Kitayama J, and Nagawa H (2004) Enhanced expression of leptin and leptin receptor (OB-R) in human breast cancer. Clin Cancer Res 10, 4325-4331.
    Pubmed CrossRef
  10. O’Brien SN, Welter BH, and Price TM (1999) Presence of leptin in breast cell lines and breast tumors. Biochem Biophys Res Commun 259, 695-698.
    Pubmed CrossRef
  11. Garofalo C, Koda M, and Cascio S (2006) Increased expression of leptin and the leptin receptor as a marker of breast cancer progression: possible role of obesity-related stimuli. Clin Cancer Res 12, 1447-1453.
    Pubmed CrossRef
  12. Guo S, Liu M, Wang G, Torroella-Kouri M, and Gonzalez-Perez RR (Array) Oncogenic role and therapeutic target of leptin signaling in breast cancer and cancer stem cells. Biochim Biophys Acta, 207-222.
    Pubmed KoreaMed
  13. Tartaglia LA, Dembski M, and Weng X (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263-1271.
    Pubmed CrossRef
  14. Perera CN, Chin HG, Duru N, and Camarillo IG (2008) Leptin-regulated gene expression in MCF-7 breast cancer cells: mechanistic insights into leptin-regulated mammary tumor growth and progression. J Endocrinol 199, 221-233.
    Pubmed CrossRef
  15. LeRoith D, and Roberts CT (2003) The insulin-like growth factor system and cancer. Cancer Lett 195, 127-137.
    Pubmed CrossRef
  16. Rajski M, Zanetti-Dallenbach R, Vogel B, Herrmann R, Rochlitz C, and Buess M (2010) IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients. BMC Med 8, 1.
    Pubmed KoreaMed CrossRef
  17. Christopoulos PF, Msaouel P, and Koutsilieris M (2015) The role of the insulin-like growth factor-1 system in breast cancer. Mol Cancer 14, 43.
    Pubmed KoreaMed CrossRef
  18. Ma J, Pollak MN, and Giovannucci E (1999) Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J Natl Cancer Inst 91, 620-625.
    Pubmed CrossRef
  19. Chan JM, Stampfer MJ, and Giovannucci E (1998) Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 279, 563-566.
    Pubmed CrossRef
  20. Endogenous H, Key TJ, Appleby PN, Reeves GK, Roddam AW, and Breast Cancer Collaborative G (2010) Insulin-like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: pooled individual data analysis of 17 prospective studies. Lancet Oncol 11, 530-542.
    Pubmed KoreaMed CrossRef
  21. Ajuwon KM, Kuske JL, and Ragland D (2003) The regulation of IGF-1 by leptin in the pig is tissue specific and independent of changes in growth hormone. J Nutr Biochem 14, 522-530.
    Pubmed CrossRef
  22. Douros JD, Baltzegar DA, and Mankiewicz J (2017) Control of leptin by metabolic state and its regulatory interactions with pituitary growth hormone and hepatic growth hormone receptors and insulin like growth factors in the tilapia (Oreochromis mossambicus). Gen Comp Endocrinol 240, 227-237.
    Pubmed KoreaMed CrossRef
  23. Daughaday WH, and Rotwein P (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10, 68-91.
    Pubmed CrossRef
  24. Tollet P, Enberg B, and Mode A (1990) Growth hormone (GH) regulation of cytochrome P-450IIC12, insulin-like growth factor-I (IGF-I), and GH receptor messenger RNA expression in primary rat hepatocytes: a hormonal interplay with insulin, IGF-I, and thyroid hormone. Mol Endocrinol 4, 1934-1942.
    Pubmed CrossRef
  25. Rogers SA, Miller SB, and Hammerman MR (1991) Insulin-like growth factor I gene expression in isolated rat renal collecting duct is stimulated by epidermal growth factor. J Clin Invest 87, 347-351.
    Pubmed KoreaMed CrossRef
  26. McCarthy TL, Centrella M, and Canalis E (1989) Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 124, 1247-1253.
    Pubmed CrossRef
  27. Jansen E, Steenbergh PH, LeRoith D, Roberts CT, and Sussenbach JS (1991) Identification of multiple transcription start sites in the human insulin-like growth factor-I gene. Mol Cell Endocrinol 78, 115-125.
    Pubmed CrossRef
  28. Angel P, and Karin M (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072, 129-157.
    Pubmed
  29. Shen Q, Uray IP, and Li Y (2008) The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors. Oncogene 27, 366-377.
    Pubmed CrossRef
  30. Catalano S, Marsico S, and Giordano C (2003) Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem 278, 28668-28676.
    Pubmed CrossRef
  31. Saxena NK, Taliaferro-Smith L, and Knight BB (2008) Bidirectional crosstalk between leptin and insulin-like growth factor-I signaling promotes invasion and migration of breast cancer cells via transactivation of epidermal growth factor receptor. Cancer Res 68, 9712-9722.
    Pubmed KoreaMed CrossRef
  32. Ahima RS, and Osei SY (2004) Leptin signaling. Physiol Behav 81, 223-241.
    Pubmed CrossRef
  33. Grothey A, Voigt W, Schober C, Muller T, Dempke W, and Schmoll HJ (1999) The role of insulin-like growth factor I and its receptor in cell growth, transformation, apoptosis, and chemoresistance in solid tumors. J Cancer Res Clin Oncol 125, 166-173.
    Pubmed CrossRef
  34. Son SW, Min BW, Lim Y, Lee YH, and Shin SY (2008) Regulatory mechanism of TNFalpha autoregulation in HaCaT cells: the role of the transcription factor EGR-1. Biochem Biophys Res Commun 374, 777-782.
    Pubmed CrossRef
  35. Shin SY, Kim JH, and Yoon H (2013) Novel antimitotic activity of 2-hydroxy-4-methoxy-2′,3′-benzochalcone (HymnPro) through the inhibition of tubulin polymerization. J Agric Food Chem 61, 12588-12597.
    Pubmed CrossRef


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