Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1α

Abstract

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1α (HIF-1α) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1α on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1α in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.

Introduction

Excess body weight has been shown to (by 30–50%) increase significantly postmenopausal breast cancer risk (Klein et al., 2002; Calle and Thun, 2004). A very recent report suggested that compared with non-obese breast cancer patients, obese subjects more frequently presented with advanced disease characterized by large, high grade, metastasizing tumors (Porter et al., 2006). Although the molecular mechanism of obesity-related mammary carcinogenesis is not clear, significant role has been suggested for cytokines produced by adipose tissue, for example, leptin.

Leptin is a pleiotropic hormone whose major function is to regulate food intake and energy balance by interacting with satiety centers in the brain (Wauters et al., 2000). Leptin also affects many peripheral organs, behaving as a mitogen, survival factor, metabolic regulator or angiogenic factor (Wauters et al., 2000). Additionally, leptin appears to promote neoplastic processes, including breast carcinogenesis (Garofalo and Surmacz, 2006; Surmacz, 2007). In breast cancer cellular models, leptin has been shown to stimulate cell growth, survival and transformation and interfere with the action of antiestrogens (Garofalo and Surmacz, 2006). In addition, leptin can modulate major pathways involved in breast cancer progression. For instance, binding of leptin to its receptor (ObR) has been shown to transactivate the Her2/neu oncogenic receptor (Eisenberg et al., 2004). Furthermore, leptin is able to induce the expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR2 in mammary tumors (Gonzalez et al., 2006). In agreement with this finding, ObR peptide antagonists can inhibit mammary tumor growth (Gonzalez et al., 2006).

Recently, others and we demonstrated that both leptin and ObR can be found in breast cancer biopsies, which suggests that leptin might affect breast cancer cells through autocrine and/or paracrine mechanisms. Importantly, the leptin system is overexpressed in breast cancer tissues, especially in high grade (G3) tumors, while it is absent or expressed at very low levels in normal mammary epithelium or benign tumors (Ishikawa et al., 2004; Garofalo et al., 2006). We also demonstrated that overexpression of leptin and ObR mRNA in breast cancer could be induced by obesity-related stimuli, such as high concentrations of insulin or estrogen (Garofalo et al., 2006). Overexpression of leptin and ObR on the protein and mRNA levels in breast cancer vs normal epithelium has been confirmed by other investigators (Ishikawa et al., 2004; Revillion et al., 2006).

The mechanism of leptin induction in breast cancer cells is unknown. Consequently, we investigated whether insulin and hypoxia-mimetic agents might regulate the expression of the leptin gene in this cell context. The human leptin gene promoter is 3000 long and contains several regulatory motifs, including AP2, SP-1, CREB, C/EBP, GRE, CRE elements (Mason et al., 1998; Ahima and Flier, 2000; Melzner et al., 2002; Meissner et al., 2003). Most importantly, the promoter contains eight hypoxia-responsive elements (HRE) with the minimal core sequence 5′-RCGTG-3′ that can recruit hypoxia-inducible factor (HIF) (Grosfeld et al., 2002; Meissner et al., 2003). HIF is a master transcriptional factor in nutrient stress signaling and can induce an array of genes involved in energy metabolism, neovascularization, survival, cell migration and pH balance. In addition, HIF can promote metastatic processes by activating tumor neoangiogenesis, and increasing cell motility and invasion (Zhong et al., 1999; Hirota and Semenza, 2006; Pouyssegur et al., 2006).

HIF is a heterodimer of a constitutively expressed HIF-1β subunit and an oxygen-regulated, unstable HIF-1α subunit. HIF activation involves HIF-1α stabilization, its nuclear translocation, heterodimerization and interaction with other transcriptional regulators, of which the most important is p300, a histone acetyl transfersase (Ebert and Bunn, 1998; Gray et al., 2005). HIF-1α expression is significantly increased under hypoxic conditions, but it can also occur in normoxia upon activation of the mTOR pathway (Pouyssegur et al., 2006). We investigated whether in breast cancer cells, insulin, a potent inducer of mTOR pathway and hypoxia-mimetic agents can stimulate leptin expression through HIF-1α.

Results

Insulin, CoCl2 and hypoxia enhance nuclear accumulation of HIF-1α in MCF-7 breast cancer cells

Previously, we reported that leptin is overexpressed in breast cancer and that this overexpression might be caused by obesity-related stimuli, such as hyperinsulinemia or hypoxia-mimetic agents (Garofalo et al., 2006). Because the leptin promoter contains multiple sites that can recruit HIF, we hypothesized that increased leptin expression might be related to accumulation of nuclear HIF-1α, resulting in increased HIF loading on leptin regulatory sequences. Thus, we assessed if insulin alone or in combination with either hypoxia-mimetic agent CoCl2 or physiological hypoxia could enhance nuclear HIF-1α expression in MCF-7 cells (Figure 1).

Figure 1
figure1

Insulin, CoCl2 and hypoxia increase nuclear hypoxia-inducible factor (HIF)-1α levels. HIF-1α expression was studied by WB, as described in ‘Materials and methods’. (a) HIF-1α expression in 100 μg of nuclear proteins obtained from cells treated for 4 h with 0–680 nM doses of insulin (Ins); (b) HIF-1α expression in 50 μg of nuclear proteins obtained from cells untreated (C), or treated for 4 h with 340 nM insulin (Ins), and/or CoCl2 and/or hypoxia (Hyp). (a and b) Protein loading and purity of fractions were controlled by re-probing WB filters for the expression of a cytoplasmic protein β-tubulin (TUB) and a nuclear marker nucleolin (NUC). Relative HIF-1α expression normalized to NUC is presented in graphs; bars represent s.e.

The levels of nuclear HIF-1α were significantly increased by insulin in a dose-dependent manner, with the maximum effects (2.5-fold increase over control) produced by the 340 nM dose (Figure 1a). CoCl2 and hypoxia treatments stimulated nuclear HIF-1α accumulation by 7- and 10-fold, respectively. The addition of insulin at 85–680 nM doses did not enhance the effects of CoCl2 or hypoxia (Figure 1b and data not shown).

Insulin, CoCl2 and hypoxia stimulate leptin mRNA expression

Next, we used quantitative real-time–PCR (QRT–PCR) to measure leptin mRNA expression under different treatments in MCF-7 cells (Figure 2). Relative to basal levels, addition of CoCl2 increased leptin mRNA by 1.8. Culturing the cells under hypoxia for 4 and 16 h stimulated leptin mRNA by 2.1- and 3.9-fold, respectively. Insulin alone was most effective in increasing leptin mRNA at 340 and 680 nM doses. Remarkably, the highest (over sixfold) leptin mRNA expression was observed under combined insulin plus CoCl2 or insulin plus hypoxia conditions (Figure 2).

Figure 2
figure2

Insulin, CoCl2 and hypoxia increase leptin mRNA expression. The abundance of leptin mRNA was studied with QRT–PCR in MCF-7 cells untreated (C) or treated for 4 h with different doses of insulin (Ins), and/or 100 μ M CoCl2 and/or hypoxia (Hyp), as described in ‘Materials and methods’. The graph represents the increase of leptin mRNA relative to the increase of α-actin mRNA in the same sample ±s.e.

Insulin, CoCl2 and hypoxia induce leptin protein expression

The expression of intracellular leptin protein was studied using immunofluorescence and deconvoluted microscopy, while the abundance of secreted leptin was measured by enzyme-linked immunosorbent assay (ELISA; Figure 3). Cellular leptin staining was detectable in 15±1% of cells treated with 340 nM insulin, 55±10% of cells under CoCl2 treatment and 67±4% of cells under hypoxia. The combined insulin plus CoCl2 or hypoxia treatments resulted in 55±6 and 61±0% cells positive for leptin staining, respectively (Figure 3). Parallel experiments revealed low HIF-1α expression in 40±4% of cells stimulated with insulin, and abundant HIF-1α levels in 62±4 and 80±7% of cells treated with CoCl2 and combined treatment, respectively (Figure 3). Under hypoxia and hypoxia plus insulin, high HIF-1α levels were detected in 80±5 and 78±7%, respectively (Figure 3).

Figure 3
figure3

Insulin, CoCl2 and hypoxia increase hypoxia-inducible factor (HIF)-1α and leptin protein expression. MCF-7 cells were treated for 16 h with 340 nM insulin (Ins), and/or CoCl2 and/or hypoxia (Hyp), as described in ‘Materials and methods’. The expression of HIF-1α (red fluorescence) and leptin (LEP, green fluorescence) was assessed by immunostaining with specific antibodies (Abs) and deconvoluted microscopy, as detailed in ‘Materials and methods’. Cell nuclei were identified with DAPI (blue fluorescence). In control experiments (Abs C), primary Abs were omitted. In parallel, the abundance of secreted leptin (pg/ml) in conditioned medium of untreated and treated cells was determined by ELISA, as described in ‘Materials and methods’.

Next, we tested the effects of various treatments on bioactive leptin secretion. Insulin alone was most effective at 340 nM dose, stimulating synthesis of 16.2±0.2 pg/ml leptin, while insulin treatment at 170 nM induced 14.2±1.0 pg/ml leptin and 680 and 1000 nM induced 12.0±0.5 and 5.0±0.1 pg/ml leptin, respectively. The greatest leptin synthesis was seen under combined stimuli (340 nM insulin plus CoCl2 or plus hypoxia) (Figure 3).

Leptin promoter is activated by insulin and hypoxia-mimetic treatment in breast cancer cells

To address the molecular mechanism by which insulin or hypoxia might activate leptin expression, we used the reporter plasmid, pGL3-OB1, containing the luciferase gene under the control of leptin regulatory sequences (−2924 to +31). Insulin and CoCl2 as well as the combination of both stimuli significantly induced luciferase activity in MCF-7 cells transfected with pGL3-OB1 relative to cells transfected with an empty vector pGL3 (Figure 4). Specifically, insulin alone increased luciferase activity by 40%, CoCl2 by 69% and the combination of both by 111%.

Figure 4
figure4

Insulin and CoCl2 stimulate leptin promoter activity in breast cancer cells. Leptin promoter activation was tested using luciferase assays, as described in ‘Materials and methods’. The cells transfected with the control plasmid (pGL3) or the leptin promoter-luciferase plasmid (OB1) were treated with 340 nM insulin (Ins), 100 μ M CoCl2 or left untreated (C). Luciferase activity was calculated as relative fold induction vs untreated cells. Bars represent s.e., asterisks (*) indicate statistically significant differences between pGL3 and OB1 transfections.

HIF-1α binds multiple HRE motifs in the leptin promoter in vitro

To address molecular mechanisms of leptin promoter activation in breast cancer cells, we assessed whether increased nuclear accumulation of HIF-1α correlates with elevated HIF-1α binding to HRE motifs. We selected three HRE-containing regions (HRE-1, proximal to ATG, HRE-2, in the middle of the leptin promoter and HRE-3, most distant from ATG). By using DNA affinity precipitation assay (DAPA), we found that CoCl2 and the combined CoCl2 plus insulin treatment increased HIF-1α binding to all three HREs (Figure 5). The greatest HIF-1α binding was seen with HRE-1 and HRE-2, and the least with HRE-3. Insulin stimulated significant HIF-1α interaction with HRE-1, but not with HRE-2 or HRE-3. Combined insulin plus CoCl2 treatments produced results statistically similar to that obtained with CoCl2 alone (Figure 5).

Figure 5
figure5

Hypoxia-inducible factor (HIF)-1α binds several hypoxia-responsive elements (HRE) motifs in the leptin promoter. The binding of HIF-1α to three different leptin promoter regions was assessed in vitro by DAPA, as described in ‘Materials and methods’. Each studied region contains one HIF motif, specifically, (−120) ACGTC in HRE-1; (−627) GCGTG in HRE-2 and (−1501) ACGTG in HRE-3. In control experiments (C), HIF-1α-HRE interactions were tested in the presence of excess unlabeled probes. The graphs represent HIF abundance on HREs under different conditions ±s.e.

CoCl2 and insulin stimulate association of the HIF-1α/p300 complex with the leptin promoter in vivo

Next, we tested using chromatin immunoprecipitation (ChIP) assays if CoCl2 and insulin stimulate HIF-1α interaction with the leptin promoter in vivo. All treatments significantly increased HIF-1α binding to the proximal leptin promoter region HRE-A. This region contains four HRE sites, including described above HRE-1 and HRE-2 as well as multiple GC-rich sequences that have been shown to improve HIF activity (Miki et al., 2004) (Figure 6). The greatest HIF-1α binding to HRE-A was detected under insulin plus CoCl2 treatments, while the lowest under insulin alone. By using reverse ChIP (re-ChIP), we demonstrated that in all cases, the HIF-1 complex interacting with HRE-A contained the major HIF coactivator p300 (Figure 6).

Figure 6
figure6

Hypoxia-inducible factor (HIF)-1α and p300 are corecruited to hypoxia-responsive elements (HRE)-containing regions in the leptin promoter. The binding of HIF-1α and corecruitment of p300 to different leptin promoter regions was tested by chromatin immunoprecipitation (ChIP) and reverse ChIP (re-ChIP) assays, as described in ‘Materials and methods’. The HRE-A region encompasses four potential HRE binding sites, including those present in HRE1 and HRE2; the HRE-B region contains two potential HIF binding sites, including that present in HRE3. DNA input in PCR reactions was assessed using HRE-A (not shown) and HRE-B primers on samples before IPs. The graph represents relative abundance of HIF-1α or p300 on HREs under different conditions ±s.e.

The HIF-1α/p300 complex was also detected on the distal HRE-B region that contains two HRE sites, but in this case the amounts of HIF-1α under all treatments were significantly lower than that seen with HRE-A. Furthermore, no synergistic effects of CoCl2 and insulin were noted (Figure 6). Neither HIF-1α nor p300 was detected on the GAPDH promoter (which does not contain HREs) under treatments used (data not shown).

CoCl2- and insulin-induced expression of leptin in breast cancer cells requires HIF-1α

The requirement for HIF-1α in the activation of leptin expression in breast cancer cells was studied using RNA interference. We found that 90% HIF-1α knockdown was paralleled by 70% inhibition of leptin mRNA synthesis (data not shown) and similar inhibition of cellular leptin expression under all treatment conditions (Figure 7).

Figure 7
figure7

Hypoxia-inducible factor (HIF)-1α knockdown reduces leptin expression. (a) MCF-7 cells were transfected with HIF-1α siRNA, as described in ‘Materials and methods’, or were left untransfected. In control experiments, the cells were transfected with lamin A/C siRNA. The expression of HIF-1α was assessed by WB in whole cell protein lysates. The expression of β-tubulin (TUB) was probed as control of protein loading. (b) The expression of HIF-1α and leptin (LEP) in MCF-7 cells transfected with HIF-1α siRNA was studied by immunofluorescence and deconvoluted microscopy as described in Figure 3 legend.

Discussion

Although recent evidence suggests that the leptin/ObR system is significantly overexpressed in breast cancers and related to tumor progression (Ishikawa et al., 2004; Garofalo et al., 2006; Revillion et al., 2006; Snoussi et al., 2006), molecular mechanisms underlying this phenomenon have not been delineated. Here, we demonstrated that leptin expression in breast cancer cells can be induced by hyperinsulinemia, hypoxia and/or hypoxia-mimetic treatments, the conditions that can be associated with obesity (Losso and Bawadi, 2005; Garofalo et al., 2006).

First, we noted that the above treatments alone or in combination increased nuclear expression of HIF-1α, the HIF subunit that is known to be stabilized by hypoxic conditions as well as by activation of the insulin-sensitive mTOR pathway (Majumder et al., 2004; Treins et al., 2005; Pore et al., 2006). In MCF-7 cells, nuclear accumulation of HIF-1α in response to insulin was dose-dependent and maximal with the 340 nM dose. The greatest nuclear HIF-1α levels under physiological hypoxia were seen at 16 h, and under 100 nM CoCl2 treatment at 4 h (Figure 1). Similar conditions stimulated maximal leptin mRNA and leptin protein expression. Notably, the highest leptin mRNA and protein expression were observed under combined insulin and hypoxia or CoCl2 treatments (Figures 2 and 3). The same conditions and with similar dynamics appeared to stimulate the leptin gene promoter (from −2924 to +31; Figure 4). Although the same promoter has previously been shown to respond to glucocorticoids in 3T3-L1 cells (De Vos et al., 1998), and to insulin and hypoxia in BeWo cells (Grosfeld et al., 2002; Meissner et al., 2003), its activity in breast cancer cells has never been described.

Because the leptin promoter contains multiple HRE motifs, we tested whether high levels of insulin and hypoxic conditions can stimulate leptin expression through HIF. We demonstrated that in MCF-7 cells nuclear HIF-1α is able to associate with several leptin promoter domains containing HREs (Figures 5 and 6). The highest HIF-1α binding was detected on the proximal promoter (HRE-A), which contains four HRE motifs as well as three GC-rich regions that can enhance HIF activity (Miki et al., 2004). We also found that the HIF-1α complex interacting with the proximal promoter included p300 histone acetyltransferase, the major HIF coactivator (Figure 6). In ChIP assays, we noted that combined insulin plus CoCl2 treatment induced slightly better HIF-1α loading on the proximal HRE-A promoter region, compared with single treatments (Figure 6). These synergistic effects were not detected using DAPA, possibly because this technique probes for protein binding to short DNA fragments and cannot fully reflect association of HIF-1α to native chromatin.

The above results are in agreement with data obtained in chorioncarcinoma cell model, where increased leptin expression was observed upon 500 nM insulin, hypoxia and combined treatments (Meissner et al., 2003). However, we are the first to show that both stimuli induce leptin in breast cancer cells acting through a common pathway involving HIF-1α. Most studies on leptin promoter activation relied on luciferase and other in vitro assays. Our report is first to demonstrate in vivo that the HIF-1α/p300 complex interacts with and activates the proximal leptin promoter in breast cancer cells. Furthermore, we established that HIF-1α is not only involved, but also required for insulin- and CoCl2-stimulated leptin expression in MCF-7 cells.

Even though HIF seems to be a major factor regulating leptin mRNA and protein expression in our cell model, we acknowledge that it cannot be the exclusive mediator of leptin expression. This is suggested by the fact that addition of insulin to hypoxia or CoCl2 increased leptin mRNA and protein synthesis relative to single treatments but did not elevate nuclear HIF-1α levels and did not increase HIF-1α loading on distant HREs (Figures 1b, 6 and 7). Furthermore, reduction of HIF-1α expression by 90% was not sufficient to produce similar inhibition of leptin expression under combined treatments (Figure 7). This suggest that leptin must be regulated by other insulin-sensitive transcriptional mechanism, as suggested before (Meissner et al., 2003). In fact, we found that in some breast cancer cells, insulin upregulates leptin expression predominantly via SP-1-dependent mechanisms (data not shown).

Cumulatively, our results indicate that one of the mechanisms of leptin overexpression in breast tumors might involve HIF-1α, a component of HIF transcriptional factor that can be upregulated by hypoxia and hyperinsulinemia. Notably, HIF-1α is often overexpressed in invasive breast cancer (Vleugel et al., 2005) and is a predictive marker of chemotherapy failure (Generali et al., 2006). Thus, molecular targeting of HIF-1α might help in the treatment of leptin-overexpressing tumors.

Materials and methods

Cell culture and treatments

MCF-7 cells were grown as described before (Garofalo et al., 2004). A total of 70% confluent cultures were treated with 340 nM insulin (Sigma, St Louis, MO, USA), 100 μ M CoCl2 (Sigma) or a combination of both stimuli. Hypoxia was accomplished by culturing the cells in a hypoxic chamber (Coy Laboratories, Grass Lake, MI, USA) with 1% O2, 94% N2, 5% CO2 at 37°C.

Western blotting

The cells were stimulated with 340 nM insulin and/or 100 μ M CoCl2 for 4 h. The expression of proteins was analysed in 50–100 μg of nuclear cell lysates obtained as described before (Morelli et al., 2004). The following antibodies (Abs) were used: HIF-1α mAb (B&D systems, Minneapolis, MN, USA), β-tubulin H235 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and nucleolin mAb (Santa Cruz Biotechnology). The intensity of bands was measured as described before (Morelli et al., 2004).

Luciferase reporter assays

MCF-7 cells were grown in 24-well plates were transfected for 6 h with 0.5 μg DNA mixture/well using Fugene 6 (Roche, Indianapolis, IN, USA). The transfection mixtures contained 0.3 μg of the leptin promoter reporter plasmid pGL3-Ob1 (or an empty vector pGL3) and 50 ng of a plasmid encoding Renilla luciferase (RI Luc) (Promega, Madison, WI, USA). The pGL3-Ob1 plasmid encodes the firefly luciferase (Luc) cDNA under the leptin promoter (from −2924 to +31) (Miller et al., 1996; De Vos et al., 1998). Upon transfection, the cells were treated with 340 nM insulin and/or 100 μ M CoCl2 for 4 h, or left untreated. Luciferase activity (Luc and RI Luc) were measured as described before (Morelli et al., 2004).

Immunofluorescence/deconvoluted microscopy

A total of 5 × 104 MCF-7 cells were plated in 2-well Permanox chamber slides (Nunc, Rockester, NY, USA). After 24 h, the cells were treated with 340 nM insulin and/or 100 μ M CoCl2, and/or hypoxia for 4 h (for HIF-1α detection) or 16 h (for leptin detection). Then the cells were washed with PBS and fixed for 20 min at 4°C in 4% paraformaldehyde for HIF-1α staining, or for 10 min at −20°C in methanol for leptin staining. Next, the cells were permeabilized with 0.2% Triton X-100, and unspecific binding was blocked in 7.5% BSA fraction V for 1 h at room temperature. HIF-1α expression was detected using 200 μg/ml HIF-1α pAb H-184 (Santa Cruz Biotechnology) and rhodamine-conjugated donkey anti-rabbit immunoglobulin G (IgG) (1:500, Santa Cruz Biotechnology). Leptin expression was probed with 200 μg/ml Ob pAb A-20 (Santa Cruz Biotechnology) and donkey anti-rabbit IgG-FITC (1:500, Santa Cruz Biotechnology). In control experiments, primary Abs were replaced by non-immune serum. The slides were covered with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) to allow visualization of cell nuclei. The abundance of nuclear HIF-1α and leptin was assessed using Olympus IX81 deconvoluted microscope and Slidebook software. HIF-1α and leptin expression were quantified by determining percentage of positive cells in at least 10 viewing fields.

ELISA

A total of 1.3 × 107 MCF-7 cells were left untreated or were treated for 16 h with 340 nM insulin, and/or 100 μ M CoCl2 and/or cultured under hypoxic conditions with or without insulin. Conditioned medium was collected and concentrated with Amicon centrifugal filter 10K (Millipore, Billerica, CA, USA) to final volume of 500 μl. Leptin abundance was measured using the ELISA low range leptin kit (R&D Systems, Minneapolis, MN, USA) following manufacturer's instructions.

Quantitative real-time–PCR

MCF-7 cells were treated for 4 h with 340 nM insulin, and/or 100 μ M CoCl2 and/or hypoxia. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). A total of 3 μg of RNA was reverse transcribed using the TaqMan RT kit (Applied Biosystems, Foster City, CA, USA). A total of 2 μl of the RT products were used to amplify leptin sequences using the Hs00174877 A1 Lep TaqMan kit (Applied Biosystems). To normalize QRT–PCR reactions, parallel TaqMan assays were run on each sample for α-actin. Leptin mRNA content relative to α-actin mRNA was determined using a comparative CT method (Applied Biosystems User Bulletin no. 2). An average CT value for each RNA was obtained for replicate reactions. Similar method was used to evaluate leptin mRNA levels in HIF-1α siRNA-treated cells.

DNA affinity precipitation assay

Binding of nuclear HIF-1α to three HRE motifs (HRE-1: region −103 to −134; HRE-2: −611 to −641 and HRE-3: −1511 to −1541) was assessed using DAPA protocol described before (Cascio et al., 2007). Briefly, 70 μg of nuclear proteins obtained from cells stimulated with 340 nM insulin and/or 100 μ M CoCl2 for 4 h were mixed with 2 μg of specific biotinylated DNA HRE probes in 400 μl of Buffer D (Cascio et al., 2007) and then precipitated with 50 μl of streptavidin-agarose beads (Invitrogen). Upon removal of beads, supernatants were analysed for the presence and abundance of HIF-1α by western blot (WB).

The specific HRE probes were prepared by annealing a biotinylated sense oligonucleotide (HRE-1, -2 and -3) with the corresponding non-biotinylated antisense oligonucleotide. The pairs were HRE-1: 5′-Bio-IndexTermCTAGCAGCCGCCCGGCACGTCGCTACCCTGA-3′ and 5′-IndexTermTCAGGGTAGCGACGTGCCGGGCGGCTGCTAG-3′; HRE-2: 5′-Bio-IndexTermTCCAGAGAGCGTGCACTCCCTGGGGTGCCA-3′ and 5′-IndexTermTGGCACCCCAGGGAGTGCACGCTCTCTGGA; HRE-3: 5′-Bio-IndexTermTATCTGGTGCCCAACGTGGGATACTGAGAT-3′ and 5′-IndexTermATCTCAGTATCCCACGTTGGGCACCAGATA. The specificity of probe–protein interactions was tested by addition of a 10-fold excess of unlabeled probes.

Chromatin immunoprecipitation and reverse ChIP

MCF-7 cells were stimulated with 340 nM insulin and/or 100 μ M CoCl2 for 4 h, or left untreated. Then, the cultures were cross-linked with 1% formaldehyde and soluble chromatin was obtained as described by us before (Morelli et al., 2004). To precipitate HIF-1α associated chromatin, we used HIF-1α pAb (Santa Cruz). A total of 5 μl sample of each HIF-1α ChIP product was used to detect specific HREs by PCR. The following primers were used: HRE-A (region from −992 to +377) forward 5′-IndexTermGCGCAGTGGGGACCAGAA-3′, reverse 5′-IndexTermCACCACCTCTGTGGAGTAG-3′; HRE-B (−1892 to −1403) forward 5′-IndexTermTTGTGGTCAGACCAGTTTTCT-3′, reverse 5′-IndexTermGTTTGGTAATGCCCAAAAGCT-3′. The PCR conditions for HRE-A region were as follows: 1 min at 94°C, 1 min at 53°C, 1 min 20 s at 72°C. For HRE-B region: 1 min 94°C, 1 min at 60°C, 1 min at 72°C. The amplification of HRE regions was analysed after PCR 32 cycles. In control samples, the primary Abs were replaced with non-immune rabbit IgG.

Re-ChIP (Morelli et al., 2004) was used to test the presence of p300 associated with HIF-1α. HIF-1α ChIP pellets obtained as described above were eluted, precipitated with p300 pAb (Upstate Biotechnology/Millipore) and processed as for one-step ChIP.

HIF1α RNA interference

HIF-1α expression was inhibited using the following siRNA oligonucleotides: HIF siRNA I 5′-IndexTermUGAGGAAGUACCAUUAUAUdTdT-3′ and HIF siRNA II 5′-IndexTermUUAUGGUUCUCACAGAUGAdTdT-3′ (Dharmacon, Lafayette, CO, USA). A total of 800 nM of HIF-1α siRNA I+II was mixed with the transfection agent RNAiFect (Qiagen, Valencia, CA, USA) (siRNA:RNAiFect ratio 1:3) and incubated for 15 min at room temperature. Then the mixture was transfected into 70% confluent cultures of MCF-7 cells for 6 h. After that, the cells were placed in fresh medium for 24 h and then treated with 340 nM insulin, and/or 100 μ M CoCl2. As controls, we used lamin A/C siRNA (Qiagen).

References

  1. Ahima RS, Flier JS . (2000). Adipose tissue as an endocrine organ. Annu Rev Physiol 62: 413–437.

  2. Calle EE, Thun MJ . (2004). Obesity and cancer. Oncogene 23: 6365–6378.

  3. Cascio S, Bartella V, Garofalo C, Russo A, Giordano A, Surmacz E . (2007). Insulin-like growth factor 1 differentially regulates estrogen receptor-dependent transcription at estrogen response element and AP-1 sites in breast cancer cells. J Biol Chem 282: 3498–3506.

  4. De Vos P, Lefebvre AM, Shrivo I, Fruchart JC, Auwerx J . (1998). Glucocorticoids induce the expression of the leptin gene through a non-classical mechanism of transcriptional activation. Eur J Biochem 253: 619–626.

  5. Ebert BL, Bunn HF . (1998). Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein. Mol Cell Biol 18: 4089–4096.

  6. Eisenberg A, Biener E, Charlier M, Krishnan RV, Djiane J, Herman B et al. (2004). Transactivation of erbB2 by short and long isoforms of leptin receptors. FEBS Lett 565: 139–142.

  7. Garofalo C, Koda M, Cascio S, Sulkowska M, Kanczuga-Koda L, Golaszewska J et al. (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.

  8. Garofalo C, Sisci D, Surmacz E . (2004). Leptin interferes with the effects of the antiestrogen ICI 182,780 in MCF-7 breast cancer cells. Clin Cancer Res 10: 6466–6475.

  9. Garofalo C, Surmacz E . (2006). Leptin and cancer. J Cell Physiol 207: 12–22.

  10. Generali D, Berruti A, Brizzi MP, Campo L, Bonardi S, Wigfield S et al. (2006). Hypoxia-inducible factor-1alpha expression predicts a poor response to primary chemoendocrine therapy and disease-free survival in primary human breast cancer. Clin Cancer Res 12: 4562–4568.

  11. Gonzalez RR, Cherfils S, Escobar M, Yoo JH, Carino C, Styer AK et al. (2006). Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2). J Biol Chem 281: 26320–26328.

  12. Gray MJ, Zhang J, Ellis LM, Semenza GL, Evans DB, Watowich SS et al. (2005). HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas. Oncogene 24: 3110–3120.

  13. Grosfeld A, Andre J, Hauguel-De Mouzon S, Berra E, Pouyssegur J, Guerre-Millo M . (2002). Hypoxia-inducible factor 1 transactivates the human leptin gene promoter. J Biol Chem 277: 42953–42957.

  14. Hirota K, Semenza GL . (2006). Regulation of angiogenesis by hypoxia-inducible factor 1. Crit Rev Oncol Hematol 59: 15–26.

  15. Ishikawa M, Kitayama J, Nagawa H . (2004). Enhanced expression of leptin and leptin receptor (OB-R) in human breast cancer. Clin Cancer Res 10: 4325–4331.

  16. Klein S, Wadden T, Sugerman HJ . (2002). AGA technical review on obesity. Gastroenterology 123: 882–932.

  17. Losso JN, Bawadi HA . (2005). Hypoxia inducible factor pathways as targets for functional foods. J Agric Food Chem 53: 3751–3768.

  18. Majumder PK, Febbo PG, Bikoff R, Berger R, Xue Q, McMahon LM et al. (2004). mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med 10: 594–601.

  19. Mason MM, He Y, Chen H, Quon MJ, Reitman M . (1998). Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor. Endocrinology 139: 1013–1022.

  20. Meissner U, Ostreicher I, Allabauer I, Rascher W, Dotsch J . (2003). Synergistic effects of hypoxia and insulin are regulated by different transcriptional elements of the human leptin promoter. Biochem Biophys Res Commun 303: 707–712.

  21. Melzner I, Scott V, Dorsch K, Fischer P, Wabitsch M, Bruderlein S et al. (2002). Leptin gene expression in human preadipocytes is switched on by maturation-induced demethylation of distinct CpGs in its proximal promoter. J Biol Chem 277: 45420–45427.

  22. Miki N, Ikuta M, Matsui T . (2004). Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1. J Biol Chem 279: 15025–15031.

  23. Miller SG, De Vos P, Guerre-Millo M, Wong K, Hermann T, Staels B et al. (1996). The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression. Proc Natl Acad Sci USA 93: 5507–5511.

  24. Morelli C, Garofalo C, Sisci D, del Rincon S, Cascio S, Tu X et al. (2004). Nuclear insulin receptor substrate 1 interacts with estrogen receptor alpha at ERE promoters. Oncogene 23: 7517–7526.

  25. Pore N, Jiang Z, Shu HK, Bernhard E, Kao GD, Maity A . (2006). Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway. Mol Cancer Res 4: 471–479.

  26. Porter GA, Inglis KM, Wood LA, Veugelers PJ . (2006). Effect of obesity on presentation of breast cancer. Ann Surg Oncol 13: 327–332.

  27. Pouyssegur J, Dayan F, Mazure NM . (2006). Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441: 437–443.

  28. Revillion F, Charlier M, Lhotellier V, Hornez L, Giard S, Baranzelli MC et al. (2006). Messenger RNA expression of leptin and leptin receptors and their prognostic value in 322 human primary breast cancers. Clin Cancer Res 12: 2088–2094.

  29. Snoussi K, Strosberg AD, Bouaouina N, Ben Ahmed S, Helal AN, Chouchane L . (2006). Leptin and leptin receptor polymorphisms are associated with increased risk and poor prognosis of breast carcinoma. BMC Cancer 6: 38.

  30. Surmacz E . (2007). Obesity hormone leptin: a new target in breast cancer? Breast Cancer Res 9: 301.

  31. Treins C, Giorgetti-Peraldi S, Murdaca J, Monthouel-Kartmann MN, Van Obberghen E . (2005). Regulation of hypoxia-inducible factor (HIF)-1 activity and expression of HIF hydroxylases in response to insulin-like growth factor I. Mol Endocrinol 19: 1304–1317.

  32. Vleugel MM, Greijer AE, Shvarts A, van der Groep P, van Berkel M, Aarbodem Y et al. (2005). Differential prognostic impact of hypoxia induced and diffuse HIF1 alpha expression in invasive breast cancer. J Clin Pathol 58: 172–177.

  33. Wauters M, Considine RV, Van Gaal LF . (2000). Human leptin: from an adipocyte hormone to an endocrine mediator. Eur J Endocrinol 143: 293–311.

  34. Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D et al. (1999). Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 59: 5830–5835.

Download references

Acknowledgements

We are grateful to Professor Auwerx, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, Illkirch, France, for providing the OB1plasmid. This work was supported by the Sbarro Health Research Organization and the WW Smith Charitable Trust.

Author information

Correspondence to E Surmacz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cascio, S., Bartella, V., Auriemma, A. et al. Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1α. Oncogene 27, 540–547 (2008). https://doi.org/10.1038/sj.onc.1210660

Download citation

Keywords

  • breast cancer
  • HIF
  • hyperinsulinemia
  • leptin

Further reading