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Winship Cancer Institute, 1701 Uppergate Drive, Clinic C, Emory University School of Medicine, Atlanta, Georgia 30322, USA
1 Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA
(Requests for offprints should be addressed to D Sharma; Email: dsharma{at}emory.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Leptin circulates as a 16 kDa protein partially bound to plasma proteins (Houseknecht et al. 1996, Sinha et al. 1996) and exerts its actions through its specific receptors present in a variety of tissues localized to the cell membrane (Bjorback et al. 1997). Leptin receptor belongs to a family of class I cytokine receptors, which typically contain a cytokine receptor homologous domain in the extracellular region (Tartaglia 1997). All six isoforms have a similar extracellular ligand-binding domain at the amino terminus but differ at the intracellular carboxy-terminal domain. While all five short isoforms have transmembrane domains, only the long form has the intracellular motifs necessary for activation of signaling pathways (Tartaglia 1997). As with other class I cytokine receptors, the leptin signaling is thought to be transmitted mainly by the JAK/STAT pathway (Bahrenberg et al. 2002, Ahima & Osei 2004). JAKs associate constitutively with conserved box 1 and 2 motifs in the intracellular domain of Ob-Rb (long isoform leptin receptor). Binding of leptin to Ob-Rb results in autophosphorylation of JAK1 and JAK2 as well as phosphorylation of the cytoplasmic domain of Ob-Rb and the downstream transcription factors STATs (Ahima & Osei 2004). The leptin signal is terminated by induction of SOCS-3 (suppressor of cytokine signaling), a member of a family of proteins which inhibits the JAK/STAT signaling cascade (Bjorbaek et al. 1999, Emilsson et al. 1999). SOCS proteins have a variable amino-terminal domain, a central SH2 domain and a carboxy-terminal domain, termed the SOCS-box motif. They are induced by cytokines and act in a negative feedback loop to inhibit the receptor. Thus overexpression of SOCS-3 inhibits leptin-mediated tyrosine phosphorylation of JAK2 (Bjorbaek et al. 1998, 1999, Emilsson et al. 1999). Whether activation of the above pathways by leptin occurs in endometrial cancer cells remains unknown.
Endometrial cancer is the most common gynecological malignancy in developed countries, with approximately 40 000 new diagnoses each year in the US alone, where obesity is also a major health concern (Abu-Abid et al. 2002, Kaaks et al. 2002, Mueck & Seeger 2004). Therefore, the effects of obesity on human endometrial cancer represent a critical intersection between these two important health problems. A case control study of endometrial cancers in Greece showed that leptin has a strong positive association with the incidence of endometrial cancer (Petridou et al. 2002). However, whether there is a direct relationship between leptin and endometrial cancer cannot be conclusively stated, as increased leptin and endometrial cancer may both be secondary consequences of obesity. Recently, circulating levels of leptin and its mRNA were measured in patients and a possible involvement of the leptin receptor in pathogenesis of endometrial cancer was proposed (Yuan et al. 2004). Considering the fundamental role of adipocytokines in endocrine-related cancer progression, the growth regulation of endometrial cancer cells by leptin might affect their malignant progression. Therefore, in the present study, we examined the expression of leptin receptors using endometrial cancer cell lines. We further investigated the effects of leptin on endometrial cancer cell proliferation and invasion.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Antibodies for phosphorylated AKT (Phospho-AKT-Ser473), AKT, phosphorylated ERK (Phospho-p44/42 MAPK-Thr202/Tyr204), ERK, phosphorylated STAT (Phospho-STAT-Tyr705) and STAT were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for short and long forms of leptin receptors Ob-R (C-20), Ob-R (B-3) and Ob-R (H-300) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA.
Cell culture, reagents and treatments
Ishikawa and ECC1, both human endometrial adenocarcinoma cell lines, are the most widely used human endometrial-derived cell culture models (for review, see Vollmer 2003). Most importantly, both ECC1 and Ishikawa cells form tumors with glandular structures if inoculated into athymic nude mice (Satyaswaroop & Tabibzadeh 1991). ECC1 and Ishikawa cells were grown in DMEM supplemented with 5% fetal bovine serum (Gemini Bioproducts, Woodland, CA, USA) and 2 µM L-glutamine (Invitrogen). For treatment, cells were seeded at a density of 1 x 106/100mm tissue culture dish. After 16 h of serum starvation, the culture media were changed to serum-free media containing treatments as indicated. Cultures were treated with human recombinant leptin (Sigma) at 100 ng/ml. In other sets of experiments, cells were treated for the indicated durations with JAK/STAT inhibitor AG490 (Calbiochem) at 100 µM, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Cell Signaling) at 10 µM, or MAPK inhibitor PD098059 (Sigma) at 10 µM.
RNA isolation and RT-PCR
Total cellular RNA was extracted using an RNeasy Mini Kit (Qiagen) and quantified by UV absorption. RNA integrity was confirmed by using formaldehyde agarose gel electrophoresis and ethidium bromide staining. cDNA was synthesized from 2.5 µg total RNA by reverse transcription at 42 °C for 1 h using a first-strand cDNA synthesis kit (Invitrogen). The synthesized cDNA was used as a template for PCR amplication. A semiquantitative PCR amplification was carried out using specific primers designed to amplify leptin receptors, Ob- Rb (long isoform) and Ob-Rt (short isoform). The primers were as follows: Ob-Rb sense 5'-TCA CCC AGT GAT TAC AAG CT-3'; Ob-Rb antisense 5'- CTG GAG AAC TCT GAT GTC CG-3'; Ob-Rt sense 5'-CAT TTT ATC CCC ATT GAG AAG TA-3'; Ob-Rt antisense 5'-CTG AAA ATT AAG TCC TTG TGC CCA G-3'. PCR generated 1071 and a 273 bp fragments of the Ob-Rb and Ob-Rt genes respectively. To ensure that amplification of these genes was within the exponential range, different numbers of cycles (25–40) were run. Finally, 30 cycles of PCR amplification was chosen. PCR conditions were 95 °C for 5 min (denaturation), 30 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min followed by 72 °C for 5 min. In addition, specific primers for 18S RNA were used as control. The primers were sense 5'-GAG GGA GCC TGA GAA ACG G-3' and antisense 5'-GTC GGG AGT GGG TAA TTT GC-3'. PCR products were resolved by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.
Cell viability assay
Cell viability assay was performed by estimating reduction of XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide), using a commercially available kit (Roche) following the manufacturer’s instructions. ECC1 and Ishikawa cells were plated in 96-well plates at an initial density of 4 x 103 cells/well for 24 h. After 16 h of serum starvation, the culture media were changed to serum-free media containing treatments as indicated. XTT labeling reagent was added to each culture well to attain a final concentration of 0.3 mg/ml. After 4 h exposure at 37 °C, absorbance was measured at 450 and 690nm using a 96-well plate reader (SPECTRAmax PLUS; Molecular Devices, CA, USA). Pilot experiments verified that the cell densities used in experiments performed were within the linear range of the XTT assay. A standard curve was prepared using cell densities from 1 x 103 to 1 x 106, and the results were calculated with respect to the number of cells.
Immunoprecipitation of Ob-Rb and Ob-Rt
For immunoprecipitation of the long and short forms of leptin receptor, 1 mg whole-cell lysates from ECC1 and Ishikawa cells were incubated with either Ob-R (C-20) (specific for the long form of leptin receptor) or Ob-R (H-300) (for both long and short forms of leptin receptor), and the mixture rotated slowly at 4 °C for 16 h. IgG was added as negative control. A total of 20 µl packed protein A/G agarose beads were added and mixture was incubated at 4 °C for 1 h with rotation. The beads were collected by gentle centrifugation and washed twice with 1.5 ml ice-cold buffer (50mM Tris–HCl (pH 7.4), 150mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1mM Na3VO4 and 1mM NaF). After the final wash, the precipitated protein–beads complexes were resuspended in SDS sample-loading buffer, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Immunodetection was performed by first blocking the membranes for 1 h in TBS buffer (20 mM Tris–HCl (pH 7.5), 137 mM NaCl, 0.05% Tween-20) containing 5% powdered milk followed by addition of the mouse monoclonal Ob-R (B-3) antibody (specific for both long and short forms of leptin receptor) in TBS and incubating for 2 h at room temperature. Specifically bound primary antibodies were detected with peroxidase-coupled secondary antibodies and developed by enhanced chemiluminescence (ECL system; Amersham Pharmacia Biotech, Arlington Heights, IL, USA) according to the manufacturer’s instructions.
Western blotting
Whole-cell lysates were prepared by scraping cells into 250 µl ice-cold modified RIPA buffer (50mM Tris–HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 1mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1mM Na3VO4 and 1mM NaF). The lysate was rotated 360° for 1 h at 4 °C followed by centrifugation at 12 000 g for 10 min at 4 °C to clear the cellular debris. Proteins were quantified using a Bradford Protein Assay Kit (Biorad). Equal amounts of proteins were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes, and Western blot analyses were performed using the previously described antibodies. Immunodetection was performed using the Amersham ECL system.
Tumor cell invasion assay
For an in vitro model system for metastasis, we performed a Matrigel invasion assay by using a Matrigel invasion chamber from BD Biocoat Cellware (San Jose, CA, USA). Cells were seeded at a density of 1 x 105 per insert and cultured overnight. After 16 h of serum starvation, the culture media were changed to serum-free media containing treatments as indicated. Triplicate wells were used for each treatment. Cells were treated with human recombinant leptin (Sigma) at 100 ng/ml. In other sets of experiments, cells were treated with JAK/STAT inhibitor AG490 (Calbiochem) at 100 µM and PI3K inhibitor LY294002 (Cell Signaling) at 10 µM along with leptin. After 24 h incubation, cells remaining above the insert membrane were removed by gentle scraping with a sterile cotton swab. Cells that had invaded through the Matrigel to the bottom of the insert were fixed in methanol for 10 min. After being washed in PBS, the cells were stained with hematoxylin–eosin. The insert was then washed in PBS and briefly air-dried and mounted. The slides were coded to prevent counting bias, and the number of invaded cells on a representative section of each membrane were counted under a light microscope. The number of invaded cells for each experimental sample represents the average of triplicate wells.
Statistical analysis
All experiments were independently performed three times in triplicate. Data were analyzed using a paired Student’s t-test. Data were considered to be statistically significant at P < 0.05. Data are expressed as means ± S.E.M.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Leptin exerts its biological functions through binding to its receptors, which mediates a downstream signal by activating multiple signaling pathways (Ahima & Osei 2004). We first examined the expression of leptin receptors in ECC1 and Ishikawa cells. The expression of leptin receptor mRNA and protein was examined using RT-PCR and Western blot analysis. A predicted PCR product of Ob-Rb (long isoform) was obtained as 1071 bp and Ob-Rt (short isoform) as 273 bp by specific primers (Fig. 1A
) in both ECC1 and Ishikawa cells. Immunoprecipitation was performed using specific antibodies Ob-R (C-20) (recognizes only the long form of the leptin receptor) and Ob-R (H-300) (recognizes both long and short forms of leptin receptor) followed by western blot analysis using mouse monoclonal Ob-R (B-3) (recognizes both long and short forms of leptin receptor). Immunoprecipitates with specific antibodies showed the presence of both long and short forms of leptin receptor in ECC1 and Ishikawa cells whereas IgG controls did not (Fig. 1B). We next examined the effect of leptin on cell proliferation. For these experiments, ECC1 and Ishikawa cells were serum starved for 16 h followed by treatment with various concentrations of recombinant human leptin for different periods of time. The effect of leptin treatment on cell proliferation was examined by the XTT assay. Leptin stimulated the growth of ECC1 and Ishikawa cells in a time- and dose-dependent manner (Fig. 1C and D). Leptin was found to be mitogenic for both ECC1 and Ishikawa cells and the effect was maximum at 100 ng/ml for 24 h of treatment. Higher concentrations of leptin (150 and 200 ng/ml) showed no further increase for 24 h post-leptin treatments. A lower concentration, 50 ng/ml, resulted in increased proliferation after 24 h in ECC1 cells and only after 48 h in Ishikawa cells.
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To gain insight into the mechanism underlying the proliferative effect of leptin on endometrial cancer cells, we next examined the changes in STAT3 phosphorylation after the treatment of cells with leptin. Previous studies have demonstrated that leptin activates JAK, which in turn phosphorylates and activates STATs in other systems (Bahrenberg et al. 2002, Ahima & Osei 2004). Total cellular proteins were extracted from cells treated with 100 ng/ml leptin for various time periods and lysates were immunoblotted with a specific phospho-tyrosine STAT3 antibody. STAT3 phosphorylation was stimulated by leptin in a time-dependent manner resulting in an increase in STAT3 phosphorylation within 15 min of treatment (Fig. 2A). Immunoblots were reprobed with antibodies against STAT3 showing that the increase in STAT3 phosphorylation was not due to the increased STAT3 protein expression (Fig. 2A). Additional studies performed to determine whether leptin treatment induced increased phosphorylation of STAT1, STAT5, p38, JNK and SAPKs showed no significant changes (data not shown).
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). Examination of cell proliferation in these treatment conditions clearly showed that blocking STAT3 phosphorylation significantly reduced the growth stimulation of endometrial cancer cells by leptin (Fig. 2C), indicating that the activation of STAT3 is essential for the cell proliferative effect of leptin in endometrial carcinoma. Effect of leptin on the activation of ERK and AKT pathways
In an effort to determine the intracellular signaling mechanisms responsible for the proliferative effect of leptin, we examined the phosphorylation of ERK and AKT after stimulating the cells with an optimum concentration of leptin for various intervals of time. An increased phosphorylation of ERK and AKT was observed within 5–15 min after leptin treatment followed by a decline (Fig. 3A and B). Leptin had no effect on total ERK and AKT protein expression levels.
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, both agents specifically inhibited the phosphorylation of ERK and AKT respectively, without affecting the expression of total protein. Significantly, leptin failed to induce ERK and AKT phosphorylation in the presence of their specific inhibitors. Examination of cell proliferation in these treatment conditions clearly showed that blocking ERK and AKT phosphorylation significantly reduced the growth stimulation of endometrial cancer cells by leptin (Fig. 4C and D). These data indicate that the activation of ERK and AKT signaling pathways is directly involved in mediating the cell proliferative effect of leptin in endometrial carcinoma.
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The previous experiments indicated that inhibition of the JAK/STAT pathway blocked leptin-mediated induction of cell proliferation (Fig. 2B and C). Also, inhibition of ERK or AKT resulted in significant reduction in endometrial cancer cell proliferation (Fig. 4). To probe the hierarchy of these events we sought to determine if specific inhibition of JAK/STAT affects the phosphorylation of ERK and AKT involved in uterine cancer cell proliferation. Treatment with the JAK/STAT inhibitor AG490 blocked the leptin-induced hyperphosphorylation of both ERK and AKT (Fig. 5A and B). Most importantly, simultaneous treatment with leptin and AG490 could not restore the level of phosphorylation as achieved by treatment with leptin alone. These data suggest that activation of JAK/STAT is upstream of the activation of ERK and AKT pathways.
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As Ob-Rb receptors are connected with several signaling pathways involved in cell proliferation, apoptosis and cancer progression, we addressed the question of whether leptin receptors may participate in the regulation of invasion in endometrial cancer progression. As shown in Fig. 6, ECC1 cells exhibited a remarkable invasion through Matrigel-coated inserts in response to 100 ng/ml leptin. In view of the critical role of PI3K in tumor invasion (Mareel & Leroy 2003), we examined the contribution of this lipid/protein kinase in the regulation of invasiveness by leptin. Treatment with PI3K inhibitor, LY294002, significantly inhibited the invasiveness induced by 100 ng/ml leptin in endometrial cancer cells. We next examined the possible contribution of the JAK/STAT tyrosine kinase as a signaling component of the Ob-Rb receptor, in the leptin-induced invasiveness of endometrial cancer cells. Similarly, the JAK/STAT inhibitor, AG490, also inhibited leptin-induced invasiveness. Our demonstration that PI3K and JAK/STAT inhibitors abrogate the leptin-induced invasion of Matrigel confirmed that the activity of these pathways is indeed a crucial component of the signaling machinery used by the leptin receptor in promoting invasiveness in endometrial carcinoma.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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However, the direct role of leptin in endometrial cancer progression and the elucidation of signaling pathways involved have never been deciphered. Thus, in the present study, the expression of leptin receptor in ECC1 and Ishikawa cells was investigated. Both short and long isoforms of leptin receptors were observed in endometrial cancer cells, suggesting that leptin may be involved in endometrial cancer. In a recent report it was shown that not all ovarian cancer cell lines respond to leptin treatment, despite the presence of its receptors (Choi et al. 2005).
In a first attempt to investigate the signaling pathways involved in leptin-mediated induction of cell growth in endometrial cancer cells, we examined the effect of leptin on the activation of the JAK/STAT pathway. Our experiments clearly showed that leptin rapidly stimulates the JAK/STAT pathway and induces the phosphorylation of ERK and AKT, thus activating two key signal-transduction pathways associated with cell growth. Inhibition of these pathways with specific chemical inhibitors not only prevents the phosphorylation of respective key signal-transduction elements, it also directly blocks the endometrial cancer cell proliferation. Also, prevention of leptin-induced activation of the JAK/STAT pathway by specific chemical inhibition in turn significantly reduced the activation of ERK and AKT pathways. Thus, we deciphered in this report the signaling pathways directly involved in the cell proliferative response to leptin in endometrial carcinoma. Also, in the present study it became clear that leptin can trigger invasion by endometrial cancer cells via a pathway involving both PI3K and JAK/STAT pathways, as pharmacological inhibition of these pathways abolished leptininduced invasiveness significantly.
These studies represent the first steps towards understanding the molecular pathway of leptin in endometrial carcinoma. AKT provides a survival signal protecting cells from apoptosis induced by various stresses (Franke et al. 1997, Kulik et al. 1997) by multiple mechanisms such as the phosphorylation of Bad, glycogen synthase-3, forkhead transcription factor and caspase-9 (Del Peso et al. 1997, Cardone et al. 1998, Pap & Cooper 1998, Brunet et al. 1999). Phosphorylation of these proteins results in inactivation of their apoptotic functions. As shown in our report, AKT phosphorylation was increased in leptin-treated human endometrial cancer cells and inhibition of PI3K with LY294002 abolished leptin-induced proliferation. LY294002 has been tested in an ectopic skin and orthotopic brain tumor model and shown to inhibit glioma tumor growth (Su et al. 2003). It has also shown efficacy against ovarian carcinoma (Hu et al. 2000). Also, more potent AKT inhibitors such as the small molecule inhibitor API-59CJ-OMe (9-methoxy-2-methylellipticinium acetate) (Jin et al. 2004) are being developed.
Our studies not only implicate the AKT pathway in endometrial cancer cell proliferation and invasion, but importantly, we report that inhibition of the JAK/STAT pathway significantly reduced the phosphorylation of AKT and ERK and proliferation mediated via these pathways, suggesting that JAK/STAT acts upstream of ERK and AKT. The main domains of STAT3 protein include the tetramerization and leucine zipper at the amino terminus, the DNA-binding domain, and the SH2 transactivation domain at the carboxy terminus. The SH2 region is responsible for the binding of STAT3 to the tyrosine phosphorylated receptors and for the dimerization necessary for DNA binding and gene expression (Bowman et al. 2000). STAT3 is activated by phosphorylation at tyrosine residue 705 leading to dimerization, nuclear translocation, recognition of STAT3-specific DNA-binding elements and activation of target gene transcription. STAT3 is frequently found to be either constitutively activated or activated in response to specific stimuli. We found that leptin activates STAT3 in endometrial cancer cells and its inhibition not only abolishes the phosphorylation of major components of the survival pathway, AKT and ERK, but also inhibits leptin-induced endometrial cancer cell proliferation. Multiple strategies have been used to block STAT activation including indirect and direct approaches. Tyrphostin AG490 has been implicated in inhibition of proliferation of human acute lymphocytic leukemia (Meydan et al. 1996) and human and mouse myeloma cells (Catlett-Falcone et al. 1999, Burdelya et al. 2002). A recent study reported that introduction of anti-sense STAT3 oligodeoxynucleotide specifically blocked expression of STAT3 mRNA in human head and neck squamous carcinoma cell lines, inhibiting proliferation (Grandis et al. 1998). Also, a small molecule inhibitor, STA21, discovered through virtual database screening, was found to inhibit human breast cancer cells expressing constitutively active STAT3 (Song et al. 2005).
In summary, our data have, for the first time, deciphered the molecular mechanisms responsible for leptin-mediated endometrial cancer cell proliferation, establishing a direct association between obesity and endometrial carcinogenesis and showing involvement of key molecules of multiple signaling pathways.
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Acknowledgements |
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N. K. Saxena, D. Sharma, X. Ding, S. Lin, F. Marra, D. Merlin, and F. A. Anania Concomitant Activation of the JAK/STAT, PI3K/AKT, and ERK Signaling Is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells Cancer Res., March 15, 2007; 67(6): 2497 - 2507. [Abstract] [Full Text] [PDF]
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