Leptin Receptor as a Potential Target to Inhibit Human Testicular Seminoma Growth
2019; Elsevier BV; Volume: 189; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2018.11.012
ISSN1525-2191
AutoresSalvatore Panza, Luca Gelsomino, Rocco Malivindi, Vittoria Rago, Ines Barone, Cinzia Giordano, Francesca Giordano, Antonella Leggio, Alessandra Comandé, Angelo Liguori, Saveria Aquila, Daniela Bonofiglio, Sebastiano Andò, Stefania Catalano,
Tópico(s)Adipose Tissue and Metabolism
ResumoAlthough in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the in vivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer. Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the in vivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer. Testicular germ cell tumors (TGCTs) are the most commonly diagnosed solid malignancies in young men (aged 18 to 35 years), representing the leading cause of cancer-related morbidity and mortality in this age group.1Ye H. Ulbright T.M. Difficult differential diagnoses in testicular pathology.Arch Pathol Lab Med. 2012; 136: 435-446Crossref PubMed Scopus (47) Google Scholar, 2Znaor A. Lortet-Tieulent J. Jemal A. Bray F. International variations and trends in testicular cancer incidence and mortality.Eur Urol. 2014; 65: 1095-1106Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar They are very heterogeneous cancers that are classified based on clinical and histologic features into seminomas and nonseminomas.3Chieffi P. 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Review of the role of leptin in the regulation of male reproductive function.Andrologia. 2018; ([Epub ahead of print])https://doi.org/10.1111/and.12965Crossref Scopus (26) Google Scholar, 31Kawwass J.F. Summer R. Kallen C.B. Direct effects of leptin and adiponectin on peripheral reproductive tissues: a critical review.Mol Hum Reprod. 2015; 21: 617-632Crossref PubMed Scopus (57) Google Scholar their expression in human testicular seminoma as well as the role of this adipokine in the development and promotion of testicular seminoma remains largely unknown. Therefore, our aim was to evaluate the expression of leptin and its receptors in human testicular seminoma and to explore the role of leptin on the growth and progression of TCam-2 cells, a well-documented experimental model for seminoma. The effects of inhibiting leptin signaling as a potential therapeutic target to reduce testicular cancer progression also was studied using both in vitro and in vivo models. The following reagents and antibodies were used: leptin from Sigma (Milan, Italy). β-Actin/Ki-67 ObR/Ob/Akt/pAktSer437 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and MAPK/signal transducer and activators of transcription 3/pMAPKThr202/Tyr204/phosphorylated signal transducer and activators of transcription 3Tyr705 from Cell Signaling Technology (Beverly, MA). Antibodies used are listed in Table 1.Table 1List of Antibodies Used for Immunofluorescence, Immunohistochemistry, and Immunoblot AnalysisAntibodiesCompanyCatalog numberDilutionKi-67Santa Cruz Biotechnologysc-78461:100 IHCObSanta Cruz Biotechnologysc-8421:500 IB1:100 IHC1:100 IFObRSanta Cruz Biotechnologysc-83911:500 IB1:100 IHC1:100 IFpAktSer473Santa Cruz Biotechnologysc-79851:500 IBAktSanta Cruz Biotechnologysc-814341:500 IBβ-ActinSanta Cruz Biotechnologysc-698791:5000 IBpSTAT3Tyr705Cell Signaling Technology9138S1:1000 IBSTAT3Cell Signaling Technology9139S1:1000 IBpMAPKThr202/Tyr204Cell Signaling Technology9101S1:1000 IBMAPKCell Signaling Technology9102S1:1000 IBIB, immunoblot; IF, immunofluorescence; IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; Ob, leptin; ObR, leptin receptor; pSTAT, phosphorylated signal transducer and activators of transcription. Open table in a new tab IB, immunoblot; IF, immunofluorescence; IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; Ob, leptin; ObR, leptin receptor; pSTAT, phosphorylated signal transducer and activators of transcription. A total of six testicular tumors and two non-neoplastic testicular tissues were analyzed in this study. Caucasian men have a greater risk of developing testicular cancer than black, Asian-American, or American-Indian men. Testicular tumors were obtained from Caucasian men with testicular germ cell tumors (seminoma) (age, 20 to 35 years) undergoing therapeutic orchidectomy at Annunziata Hospital (Cosenza, Italy). Non-neoplastic testicular tissues were obtained from two Caucasian men (aged 29 and 32 years) with testes with a granulomatous lesion collected from the same hospital. Tissue specimens were obtained from patients who signed informed consent at Annunziata Hospital. Fresh surgical tissues were formalin-fixed/paraffin-embedded. Obtained sections were used for morphologic analyses by hematoxylin and eosin staining and immunohistochemical analyses. The clinical investigation was approved by the Ethics and Institutional Human Subject Committees at Annunziata Hospital. Paraffin-embedded sections (5-μm thick) were mounted on polylysine-precoated slides, and then deparaffinized and dehydrated (seven to eight serial sections). Immunohistochemical experiments were performed after heat-mediated antigen retrieval. Hydrogen peroxide (3% in distilled water) was used for 30 minutes to block endogenous peroxidase activity, and normal goat serum (10%) was used for 30 minutes to prevent nonspecific binding. Immunodetection was performed using anti-Ob, anti-ObR, and anti–Ki-67 primary antibodies at 4°C overnight, dilutions used are reported in Table 1. Then, biotinylated goat–anti-rabbit and rabbit–anti-goat IgG were applied for 1 hour at room temperature, followed by the avidin-biotin–horseradish peroxidase complex (Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized using diaminobenzidine chromogen (Zymed Laboratories, South San Francisco, CA). In negative control sections normal serum was used instead of the primary antibody. The immunostained slides of tumor samples were visualized using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and the images were taken with CSV1.14 software (Olympus), using a CAM XC-30 (Olympus) for image acquisition. Immunoreactivity was evaluated by a pathologist in a blinded fashion (V.R.) and scored as: 0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive; and 4, very strongly positive. The human TCam-2 seminoma cell line was a gift from Dr. Leendert H. J. Looijenga (Department of Pathology, Medical Center Rotterdam, Josephine Nefkens Institute, Rotterdam, the Netherlands), and was grown in RPMI-1640 plus 10% fetal bovine serum, 1% penicillin/streptomycin, and 200 mmol/L glutamine, at 37°C in a humidified cell culture incubator with 5% carbon dioxide. Mizuno et al32Mizuno Y. Gotoh A. Kamidono S. Kitazawa S. Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2).Nihon Hinyokika Gakkai Zasshi. 1993; 84: 1211-1218PubMed Google Scholar reported isolation and characterization of TCam-2 cells, which then were indicated, using a multidisciplinary approach as representative for human seminoma.33Russell S.M. Lechner M.G. Mokashi A. Megiel C. Jang J.K. Taylor C.R. Looijenga L.H. French C.A. Epstein A.L. Establishment and characterization of a new human extragonadal germ cell line, SEM-1, and its comparison with TCam-2 and JKT-1.Urology. 2013; 81: 464.e1-464.e9Abstract Full Text Full Text PDF Scopus (12) Google Scholar, 34de Jong J. Stoop H. Gillis A.J. Hersmus R. van Gurp R.J. van de Geijn G.J. van Drunen E. Beverloo H.B. Schneider D.T. Sherlock J.K. Baeten J. Kitazawa S. van Zoelen E.J. van Roozendaal K. Oosterhuis J.W. Looijenga L.H. Further characterization of the first seminoma cell line TCam-2.Genes Chromosomes Cancer. 2008; 47: 185-196Crossref PubMed Scopus (110) Google Scholar, 35Eckert D. Nettersheim D. Heukamp L.C. Kitazawa S. Biermann K. Schorle H. TCam-2 but not JKT-1 cells resemble seminoma in cell culture.Cell Tissue Res. 2008; 331: 529-538Crossref PubMed Scopus (63) Google Scholar MCF-7 breast cancer cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 1% l-glutamine, 1% Eagle's nonessential amino acids, and 1 mg/mL penicillin-streptomycin at 37°C with 5% CO2 air. TCam-2 cells routinely were authenticated by evaluating the expression of the well-known specific biomarkers for seminoma cells as reported by Russel et al.33Russell S.M. Lechner M.G. Mokashi A. Megiel C. Jang J.K. Taylor C.R. Looijenga L.H. French C.A. Epstein A.L. Establishment and characterization of a new human extragonadal germ cell line, SEM-1, and its comparison with TCam-2 and JKT-1.Urology. 2013; 81: 464.e1-464.e9Abstract Full Text Full Text PDF Scopus (12) Google Scholar The MCF-7 cell line was authenticated by short tandem repeats analysis at the Sequencing Core at University of Calabria (Rende, Italy). Total RNA from TCam-2 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and gene expression was evaluated by real-time RT-PCR, using SYBR Green Universal PCR Master Mix (Bio-Rad, Segrate, Italy) as previously described.36Catalano S. Panza S. Malivindi R. Giordano C. Barone I. Bossi G. Lanzino M. Sirianni R. Mauro L. Sisci D. Bonofiglio D. Andò S. Inhibition of Leydig tumor growth by farnesoid X receptor activation: the in vitro and in vivo basis for a novel therapeutic strategy.Int J Cancer. 2013; 132: 2237-2247Crossref PubMed Scopus (24) Google Scholar Each sample was normalized on GAPDH mRNA content. The relative gene expression levels were calculated as described.37Sirianni R. Chimento A. Malivindi R. Mazzitelli I. Andò S. Pezzi V. Insulin-like growth factor-I, regulating aromatase expression through steroidogenic factor 1, supports estrogen-dependent tumor Leydig cell proliferation.Cancer Res. 2007; 67: 8368-8377Crossref PubMed Scopus (68) Google Scholar Primers are listed in Table 2.Table 2Oligonucleotide Primers Used in This StudyGene nameGene symbolPrimer sequencesLeptinLEPF: 5′-GTGCGGATTCTTGTGGCTTT-3′R: 5′-GGAATGAAGTCCAAACCGGTG-3′Long-form leptin receptorOBRLF: 5′-TCACTCCGAAAGCAACAGTG-3′R: 5′-CTTTTGCCTGCTGGACTCTC-3′Short-form leptin receptorOBRSF: 5′-ATTGTGCCAGTAATTATTTCCTCTTCC-3′R: 5′-CCACCATATGTTAACTCTCAGAAGTTCAA-3′Matrix metalloproteinase-2MMP2F: 5′-TGCTGCACCTGGGATTAAGG-3′R: 5′-CACCTTTTGCTCCACGTGC-3′Matrix metalloproteinase-9MMP9F: 5′-AGTTCCCGGAGTGAGTTGAA-3′R: 5′-CTCCACTCCTCCCTTTCCTC-3′AromataseCYP19A1F: 5′-ACCCTTCTGCGTCGTGTCA-3′R: 5′-TCTGTGGAAATCCTGCGTCTT-3′Cyclin D1CCND1F: 5′-GATGCCAACCTCCTCAACGAC-3′R: 5′-CTCCTCGCACTTCTGTTCCTC-3′Heat shock protein 90HSP90AF: 5′-GGGTAAAAGTAGAGAGAAGGATCAAGGA-3′R: 5′-TGCATTTAATCCATCCAACTGAA-3′Vascular endothelial growth factorVEGFF: 5′-CCTGGTGGACATCTTCCAGGA-3′R: 5′-CTCACCGCCTCGGCTTGTCAC-3′Hypoxia-inducible factor 1-αHIF1AF: 5′-TGCACAGGCCACATTCACGT-3′R: 5′-GTTCACAAATCAGCACCAAGC-3′SurvivinBIRC5F: 5′-GGACCACCGCATCTCTACAT-3′R: 5′-GTTGCGCTTTCCTTTCTGTC-3′Glyceraldehyde 3-phosphate dehydrogenaseGAPDHF: 5′-CCCACTCCTCCACCTTTGAC-3′R: 5′-TGTTGCTAGCCAAATTCGTT-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Cell extracts were resolved by SDS-PAGE as described.38Catalano S. Malivindi R. Giordano C. Gu G. Panza S. Bonofiglio D. Lanzino M. Sisci D. Panno M.L. Andò S. Farnesoid X receptor, through the binding with steroidogenic factor 1-responsive element, inhibits aromatase expression in tumor Leydig cells.J Biol Chem. 2010; 285: 5581-5593Crossref PubMed Scopus (53) Google Scholar Blots are representative of three separate experiments. The bands of interest were quantified by the Scion Image laser densitometry scanning program (ImageJ version 1.51q; NIH, Bethesda, MD) and SDs along with associated P values for the replicates were calculated by GraphPad Prism 4 software (GraphPad, Inc., San Diego, CA). Immunofluorescence microscopy analysis was conducted as described.39Panza S. Malivindi R. Chemi F. Rago V. Giordano C. Barone I. Bonofiglio D. Gelsomino L. Giordano F. Andò S. Catalano S. Glucocorticoid receptor as a potential target to decrease aromatase expression and inhibit Leydig tumor growth.Am J Pathol. 2016; 186: 1328-1339Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with phosphate-buffered saline 0.2% Triton X-100 (Sigma), blocked with 5% bovine serum albumin (1 hour at room temperature), and incubated with anti-Ob and anti-ObR primary antibody (4°C, overnight; dilutions used are listed in Table 1), followed by fluorescein isothiocyanate–conjugated secondary antibody (30 minutes at room temperature). IgG primary antibody was used as negative control. DAPI (Sigma) staining was used for nuclei detection. Fluorescence was captured with an Olympus BX51 microscope (100× objective). Leptin levels were detected using the Enzyme-Linked Immunosorbent Assay kit (Labor Diagnostika Nord, Nordhorn, Germany) according to the manufacturer's instructions. Results are presented as picograms per milligram of protein. TCam-2 cells were treated with leptin with/without peptide Leu-Asp-Phe-Ile (LDFI) synthesized by solid-phase methodologies40Di Gioia M.L. Leggio A. Liguori A. Perri F. Solid-phase synthesis of N-Nosyl- and N-Fmoc-N-methyl-α-amino acids.J Org Chem. 2007; 72: 3723-3728Crossref PubMed Scopus (20) Google Scholar for 24 hours. For the last 6 hours, [3H]thymidine (1 μCi/mL) was added to the culture medium. At the end of incubation, cells were washed once with 10% and three times with 5% trichloroacetic acid, lyzed in 0.1 N NaOH for 30 minutes at 37°C. [3H]thymidine incorporation was determined by scintillation counting. To perform cell-cycle analysis, cells were collected by trypsinization, fixed, and stained with 100 μg/mL propidium iodide after treatment with 20 μg/mL RNase A. The DNA content was determined with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and the data were acquired with CellQuest software version 3.3 (BD Biosciences, San Jose, CA). Cell-cycle profiles were analyzed with ModFit LT version version 3.3 (BD Biosciences). Cell migration was monitored and the rate of wound healing was quantified as reported.41Giordano C. Barone I. Vircillo V. Panza S. Malivindi R. Gelsomino L. Pellegrino M. Rago V. Mauro L. Lanzino M. Panno M.L. Bonofiglio D. Catalano S. Andò S. Activated FXR inhibits leptin signaling and counteracts tumor-promoting activities of cancer-associated fibroblasts in breast malignancy.Sci Rep. 2016; 6: 21782Crossref PubMed Scopus (43) Google Scholar Briefly, cell monolayers were scraped and subjected to the different experimental conditions as indicated. Images are representative of three independent experiments (10× magnification). The rate of wound healing was quantified from the images using Scion Image and Adobe Photoshop software CC 2015 (Adobe Systems Incorporated, San Jose, CA), and SDs along with associated P values for the replicates were calculated by GraphPad Prism 4 software (GraphPad, Inc.). Cells treated with leptin with/without peptide LDFI were placed in the upper compartments of a Boyden chamber (8-μm membranes; Corning Costar, Bedford, MA). The bottom well contained regular full media. After 6 hours, migrated cells were fixed and stained with Coomassie brilliant blue (Sigma). Migration was quantified by viewing five separate fields per membrane at 20× magnification and expressed as the mean number of migrated cells. Data represent three independent experiments, performed in triplicate. The in vivo experiments were performed in 30-day-old male nude mice (nu/nu Swiss; Charles River, Milan, Italy) maintained in a sterile environment. At day 0, TCam-2 cells (1.0 × 106 cells per mouse) were inoculated into the intrascapular region, in 0.1 mL of Matrigel (Corning Costar). When the mean tumor size reached approximatively 100 mm3 of volume the mice were randomized into two groups (5 mice/group). The mice then were treated with LDFI–polyethylene glycol (PEG) (10 mg/kg/day) diluted in saline 0.3% bovine serum albumin or saline 0.3% bovine serum albumin only (vehicle) by i.p. injection. The treatment was delivered 5 days a week. Tumor development was followed twice a week by caliper measurements along two orthogonal axes: length (L) and width (W). The volume (V) of tumors was estimated by the following formula: V = L (W2)/2. Relative tumor volume (RTV) was calculated from the following formula: RTV = (Vx/V1), where Vx is the tumor volume on day X and V1 is the tumor volume at initiation of the treatment. The growth curve was obtained by plotting the mean volume of RTV on y axis against time (x axis expressed as days after starting treatment). Antitumor activity was evaluated according to tumor growth inhibition, calculated from the following formula: percentage growth inhibition = 100 − (RTVt/RTVc) × 100, where RTVt is the medium RTV of treated mice and RTVc is the median RTV of controls, both at a given time point when the antitumor effect was optimal. At the time of sacrifice, tumors were dissected out from the neighboring connective tissue, frozen, and stored in nitrogen for further analysis. The remaining tumor tissues from each sample were fixed in 4% paraformaldehyde and embedded in paraffin for histologic analyses. All animals were maintained and handled in accordance with the recommendation of the Guidelines for the Care and Use of Laboratory Animal
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