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Chemokine (C-C Motif) Ligand 1 Derived from Tumor-Associated Macrophages Contributes to Esophageal Squamous Cell Carcinoma Progression via CCR8-Mediated Akt/Proline-Rich Akt Substrate of 40 kDa/Mammalian Target of Rapamycin Pathway

2021; Elsevier BV; Volume: 191; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2021.01.004

ISSN

1525-2191

Autores

Masataka Fujikawa, Yu‐ichiro Koma, Masayoshi Hosono, Naoki Urakawa, Kohei Tanigawa, Masaki Shimizu, Takayuki Kodama, Hiroki Sakamoto, Mari Nishio, Manabu Shigeoka, Yoshihiro Kakeji, Hiroshi Yokozaki,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Tumor-associated macrophages (TAMs) promote tumor progression. The number of infiltrating TAMs is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) patients; however, the mechanism underlying this phenomenon is unclear. cDNA microarray analysis indicates that the expression of chemokine (C-C motif) ligand 1 (CCL1) is up-regulated in peripheral blood monocyte–derived macrophages stimulated using conditioned media from ESCC cells (TAM-like macrophages). Here, we evaluated the role of CCL1 in ESCC progression. CCL1 was overexpressed in TAM-like macrophages, and CCR8, a CCL1 receptor, was expressed on ESCC cell surface. TAM-like macrophages significantly enhanced the motility of ESCC cells, and neutralizing antibodies against CCL1 or CCR8 suppressed this increased motility. Recombinant human CCL1 promoted ESCC cell motility via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway. Phosphatidylinositol 3-kinase or Akt inhibitors, CCR8 silencing, and neutralizing antibody against CCR8 could significantly suppress these effects. The overexpression of CCL1 in stromal cells or CCR8 in ESCC cells was significantly associated with poor overall survival (P = 0.002 or P = 0.009, respectively) and disease-free survival (P = 0.009 or P = 0.047, respectively) in patients with ESCC. These results indicate that the interaction between stromal CCL1 and CCR8 on cancer cells promotes ESCC progression via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway, thereby providing novel therapeutic targets. Tumor-associated macrophages (TAMs) promote tumor progression. The number of infiltrating TAMs is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) patients; however, the mechanism underlying this phenomenon is unclear. cDNA microarray analysis indicates that the expression of chemokine (C-C motif) ligand 1 (CCL1) is up-regulated in peripheral blood monocyte–derived macrophages stimulated using conditioned media from ESCC cells (TAM-like macrophages). Here, we evaluated the role of CCL1 in ESCC progression. CCL1 was overexpressed in TAM-like macrophages, and CCR8, a CCL1 receptor, was expressed on ESCC cell surface. TAM-like macrophages significantly enhanced the motility of ESCC cells, and neutralizing antibodies against CCL1 or CCR8 suppressed this increased motility. Recombinant human CCL1 promoted ESCC cell motility via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway. Phosphatidylinositol 3-kinase or Akt inhibitors, CCR8 silencing, and neutralizing antibody against CCR8 could significantly suppress these effects. The overexpression of CCL1 in stromal cells or CCR8 in ESCC cells was significantly associated with poor overall survival (P = 0.002 or P = 0.009, respectively) and disease-free survival (P = 0.009 or P = 0.047, respectively) in patients with ESCC. These results indicate that the interaction between stromal CCL1 and CCR8 on cancer cells promotes ESCC progression via the Akt/proline-rich Akt substrate of 40 kDa/mammalian target of rapamycin pathway, thereby providing novel therapeutic targets. Esophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related deaths, accounting for an estimated 572,000 new cases worldwide and approximately 509,000 deaths in 2018.1Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. 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O'Brien S.J. Krangel M.S. Sequence and chromosomal location of the I-309 gene: relationship to genes encoding a family of inflammatory cytokines.J Immunol. 1990; 145: 2737-2744PubMed Google Scholar,28Miller M.D. Krangel M.S. The human cytokine I-309 is a monocyte chemoattractant.Proc Natl Acad Sci U S A. 1992; 89: 2950-2954Crossref PubMed Scopus (124) Google Scholar and selectively interacts with CCR8.29Tiffany H.L. Lautens L.L. Gao J.L. Pease J. Locati M. Combadiere C. Modi W. Bonner T.I. Murphy P.M. Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.J Exp Med. 1997; 186: 165-170Crossref PubMed Scopus (182) Google Scholar CCR8 is a member of the β chemokine receptor family and is a seven-transmembrane protein, similar to G-protein–coupled receptors.30de Munnik S.M. Smit M.J. Leurs R. Vischer H.F. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors.Front Pharmacol. 2015; 6: 40Crossref PubMed Scopus (32) Google Scholar Type 2 helper T cells, Tregs, and monocytes express CCR8.31McCully M.L. Moser B. The human cutaneous chemokine system.Front Immunol. 2011; 2: 33Crossref PubMed Scopus (23) Google Scholar The CCL1-CCR8 axis promotes the recruitment and activation of T cells in inflammatory conditions, such as dextran sulfate sodium–induced colitis,32Yang R. Liao Y. Wang L. He P. Hu Y. Yuan D. Wu Z. Sun X. Exosomes derived from M2b macrophages attenuate DSS-induced colitis.Front Immunol. 2019; 10: 2346Crossref PubMed Scopus (59) Google Scholar asthma,33Montes-Vizuet R. CC chemokine ligand 1 is released into the airways of atopic asthmatics.Eur Respir J. 2006; 28: 59-67Crossref PubMed Scopus (44) Google Scholar and atopic dermatitis.34Gombert M. Dieu-Nosjean M.C. Winterberg F. Bunemann E. Kubitza R.C. Da Cunha L. Haahtela A. Lehtimaki S. Muller A. Rieker J. Meller S. Pivarcsi A. Koreck A. Fridman W.H. Zentgraf H.W. Pavenstadt H. Amara A. Caux C. Kemeny L. Alenius H. Lauerma A. Ruzicka T. Zlotnik A. Homey B. CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation.J Immunol. 2005; 174: 5082-5091Crossref PubMed Scopus (169) Google Scholar The CCL1-CCR8 axis also contributes to tumor progression in breast cancer, melanoma, bladder cancer, and renal cancer.35Das S. Sarrou E. Podgrabinska S. Cassella M. Mungamuri S.K. Feirt N. Gordon R. Nagi C.S. Wang Y. Entenberg D. Condeelis J. Skobe M. Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses.J Exp Med. 2013; 210: 1509-1528Crossref PubMed Scopus (123) Google Scholar,36Eruslanov E. Stoffs T. Kim W.J. Daurkin I. Gilbert S.M. Su L.M. Vieweg J. Daaka Y. Kusmartsev S. Expansion of CCR8(+) inflammatory myeloid cells in cancer patients with urothelial and renal carcinomas.Clin Cancer Res. 2013; 19: 1670-1680Crossref PubMed Scopus (43) Google Scholar However, the role of the CCL1-CCR8 axis in ESCC progression has not been established. The aim of this study was to determine the role of TAM-derived CCL1 in the ESCC microenvironment. Three ESCC cell lines (TE-8, TE-9, and TE-15) were obtained from the RIKEN BioResource Center (Tsukuba, Japan). TE-8 cells are moderately differentiated ESCC cells; TE-9 cells are poorly differentiated ESCC cells; and TE-15 cells are well-differentiated ESCC cells.37Nishihira T. Hashimoto Y. Katayama M. Mori S. Kuroki T. Molecular and cellular features of esophageal cancer cells.J Cancer Res Clin Oncol. 1993; 119: 441-449Crossref PubMed Scopus (212) Google Scholar ESCC cell lines with different degrees of differentiation were selected to demonstrate that all of the in vitro results were applicable to various types of ESCC. Cells were authenticated using short tandem repeat analysis at RIKEN and at the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan) in 2009 and 2010. TE-8, TE-9, and TE-15 cells were confirmed to be mycoplasma-negative using a Venor Gem Classic Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). TE-8, TE-9, and TE-15 cells were maintained in RPMI 1640 (Wako, Osaka, Japan) medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA). CM of TE-8, TE-9, and TE-15 cells was prepared by seeding 5 × 106 tumor cells per 10 mL of complete medium in 100-mm dishes for 24 hours, and the medium was replaced with complete Dulbecco's modified Eagle's medium (Wako) supplemented with 10% human AB serum (Lonza, Walkersville, MD). After incubation for 48 hours, the supernatants were harvested, centrifuged, and stored in aliquots at −80°C. Peripheral blood mononuclear cells were collected from healthy volunteer donors after obtaining informed consent. The autoMACS Pro Separator (Miltenyi Biotec, Bergish Gladbach, Germany) was used to separate CD14+ PBMos from peripheral blood mononuclear cells based on positive selection. PBMos were incubated with macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN; 25 ng/mL) for six days to induce the formation of PBMo-derived macrophages and cultured for two days with 50% CM of TE-8, TE-9, and TE-15 cells to achieve TAM-like polarization, as demonstrated in our previous studies.21Shigeoka M. Urakawa N. Nakamura T. Nishio M. Watajima T. Kuroda D. Komori T. Kakeji Y. Semba S. Yokozaki H. Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma.Cancer Sci. 2013; 104: 1112-1119Crossref PubMed Scopus (137) Google Scholar PBMo-derived macrophages stimulated with CM of TE-8, TE-9, or TE-15 cells were defined as TAM-like PBMo-derived macrophages (TAM8, TAM9, or TAM15, respectively). A total of 69 human ESCC tissue samples, which were surgically removed at Kobe University Hospital (Kobe, Japan) from 2005 to 2010, were used in this study. None of the patients had received adjuvant chemotherapy or radiotherapy before surgery. Informed consent for the use of tissue samples was obtained from all patients, and the study was approved by the Institutional Review Board of Kobe University. All specimens were fixed with 10% formalin and embedded in paraffin wax. Histologic and clinicopathologic parameters were analyzed using the Japanese Classification of Esophageal Cancer, proposed by the Japan Esophageal Society,38Japan Esophageal SocietyJapanese Classification of Esophageal Cancer.ed 10. Kanehara & Co, Tokyo, Japan2008Google Scholar and the TNM classification, proposed by the Union for International Cancer Control.39Sobin L.H. Gospodarowicz M.K. Wittekind C. TNM Classification of Malignant Tumours. ed 7. Wiley-Blackwell, Hoboken, NJ2011Google Scholar Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The expression of CCR8 and the internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was evaluated using RT-PCR. The PCR products were subjected to electrophoresis on a 2% agarose gel. Quantitative real-time PCR for CCL1 and GAPDH was performed using the ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA). The CT values were determined by plotting the observed fluorescence against the cycle number. CT values were analyzed using the comparative CT method and normalized to those of GAPDH. Relative gene expression was estimated using the following formula: relative expression = 2–[CT(target gene) – CT(GAPDH)]. The primers were designed as follows: GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-T-CC-ACCCTGTTGCTGTA-3′ (reverse); CCL1, 5′-GGAAGAT-GTGGACAGCAAGAGC-3′ (forward) and 5′-TGTA-GGG-CTGGTAGTTTCGG-3′ (reverse); and CCR8, 5′-GT-GTG-ACAACAGTGACCGACT-3′ (forward) and 5′-CTT-CTTG-CAGACCACAAGGAC-3′ (reverse). Cells were lysed on ice using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA) containing 1% protease inhibitor and 1% phosphatase inhibitor cocktail (Sigma-Aldrich). The resulting lysates were separated onto 5% to 20% SDS polyacrylamide gels and transferred to a membrane using iBlot Gel Transfer Stack (Invitrogen, Carlsbad, CA). The membrane was blocked with 5% skim milk and incubated with primary and secondary antibodies. The protein bands were detected with ImmunoStar Reagents (Wako). The following primary antibodies were used: rabbit antibody against CCR8 (1:300; number ab8019; Abcam, Cambridge, UK), rabbit antibody against Akt (1:500; number 9272; Cell Signaling Technology, Beverly, MA), rabbit antibody against phosphorylated Akt (Ser473; 1:300; number 4060; Cell Signaling Technology), rabbit antibody against phosphorylated Akt (Thr308; 1:300; number 2965; Cell Signaling Technology), rabbit antibody against proline-rich Akt substrate of 40 kDa (PRAS40; 1:500; number 2961; Cell Signaling Technology), rabbit antibody against phosphorylated PRAS40 (Thr246; 1:300; number 13175; Cell Signaling Technology), rabbit antibody against mammalian target of rapamycin (mTOR; 1:200; number 2972; Cell Signaling Technology), rabbit antibody against phosphorylated mTOR (Ser2448; 1:200; number 2971; Cell Signaling Technology), and rabbit antibody against β-actin (1:1000; number 4970; Cell Signaling Technology). Horseradish peroxidase–linked donkey anti-rabbit IgG (number NA934V; GE Healthcare Life Science, Little Chalfont, UK) was used as the secondary antibody. TE-8, TE-9, or TE-15 cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured in serum-free RPMI 1640 medium for the survival assay. Cells were seeded at a density of 5 × 103 cells per well and cultured in 1% FBS at 37°C for the growth assay. They were treated with 0 or 10 ng/mL recombinant human CCL1 (rhCCL1; R&D Systems). After 0, 24, 48, or 96 hours, CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) was added. Absorbance was measured using a microplate reader (Infinite 200 PRO; Tecan, Mannedorf, Switzerland) at 492 nm. For the migration assay, TE-8, TE-9, or TE-15 cells (1 × 105 cells per well) in RPMI 1640 medium supplemented with 0.1% FBS were seeded in the upper chamber containing an 8-μm pore filter (BD Falcon, Lincoln Park, NY) in 24-well plates. For the invasion assay, TE-8, TE-9, or TE-15 cells (2 × 105 cells per well) in RPMI 1640 medium supplemented with 0.1% FBS were seeded in the upper chamber of the Corning BioCoat Matrigel Invasion Chamber (Corning, Tewksbury, MA) in 24-well plates. RPMI 1640 medium containing 0.1% FBS was added to the lower chamber. Cells in the upper chamber were treated with phosphatidylinositol 3-kinase inhibitor (LY294002; 1 μmol/L; Abcam), Akt inhibitor (GSK690693; 1 μmol/L; Sigma-Aldrich), or neutralizing antibody against CCR8 (10 ng/mL). rhCCL1 (10 ng/mL) or a neutralizing antibody against CCL1 (0.2 μg/mL) was added to the media in the lower chamber. After incubation at 37°C in a CO2 incubator for 24 hours (migration assay) or 48 hours (invasion assay), the cells remaining on the surface of the membrane in the upper chamber were removed using a cotton swab. Cells on the lower surface of the membrane were stained using the Diff-Quik Kit (Sysmex, Kobe, Japan). Four images at ×100 magnification were obtained from each membrane using a charge-coupled device camera, and cells were counted. The following neutralizing antibodies were used: mouse antibody against CCL1 (number MAB272; R&D Systems) and rat antibody against CCR8 (number MAB1429; R&D Systems); the following antibodies were used as the negative control: normal mouse IgG (number ab188776; Abcam) and normal rat IgG (number MAB0061; R&D Systems), respectively. PBMos (1 × 105 cells per well) were seeded in the lower chamber in 24-well plates, and stimulated using macrophage colony-stimulating factor (25 ng/mL; R&D Systems) for six days to induce the formation of macrophages, followed by incubation with 50% CM of TE-8, TE-9, and TE-15 cells to induce the formation of TAM-like macrophages. After two days, the media were replaced with RPMI 1640 medium containing 0.1% FBS. TE-8, TE-9, or TE-15 cells (migration assay, 1 × 105 cells per well; invasion assay, 2 × 105 cells per well) in RPMI 1640 medium supplemented with 0.1% FBS were seeded in the upper chamber. Transwell migration and invasion assays were performed as described above. PBMo-derived macrophages, TAM-like macrophages, and TE-8, TE-9, and TE-15 cells were cultured in 6-well plates (5 × 105 cells per well) using RPMI 1640 medium containing 10% FBS. After 48 hours, the supernatants were harvested, and CCL1 levels were determined using the Human I-309 ELISA Kit (CCL1) (number ab100536; Abcam), according to the manufacturer's instructions. The OD of each well was measured using the Infinite 200 PRO Microplate Reader at 492 nm. The CCL1 concentration in each sample was calculated using a standard curve. TE-8 cells were seeded in 60-mm dishes (5 × 105 cells per dish), and cultured in serum-free RPMI 1640 medium. After 24 hours, cells were incubated with or without rhCCL1 (10 ng/mL) for 10 minutes, and harvested for protein extraction. The proteins were analyzed using the Proteome Profiler Human Phospho-Kinase Array Kit (ARY003B; R&D Systems), according to the manufacturer's instructions. TE-8, TE-9, and TE-15 cells were transfected with 20 nmol/L siRNAs targeting CCR8 (siCCR8; Sigma-Aldrich) using Lipofectamine RNAiMAX (Invitrogen). Cells transfected with control siRNA (siNC; Sigma-Aldrich) were used as the negative control. Immunohistochemistry was performed using EnVision Dual Link System-HRP and 3,3′-diaminobenzidine (Dako Cytomation, Glostrup, Denmark). The following antibodies were used for antigen detection in ESCC tissues: rabbit antibody against CCL1 (1:50; number HPA049861; Atlas Antibody, Stockholm, Sweden) and rabbit antibody against CCR8 (1:100; number NBP2-15768; Novus Biologicals, Littleton, CO). CCL1 expression in the stroma around the cancer nest was investigated at a magnification of ×100 (four images) using a charge-coupled device camera, and CCL1-positive stromal cells were counted. The median cell counts in the cancer stroma were obtained and samples were stratified into two groups (low and high). CCR8 expression in the cancer nest was evaluated, and samples were stratified into two groups (negative and positive) based on the presence or absence of CCR8-positive cancer cells in the cancer nest. PBMo-derived macrophages, TAM-like macrophages, and TE-8, TE-9, and TE-15 cells were seeded on coverslips, and fixed with 4% paraformaldehyde in phosphate buffer solution (Wako). PBMo-derived macrophages and TAM-like macrophages were incubated with rabbit antibody against CCL1 (1:50; number HPA049861; Atlas Antibody) and mouse antibody against CD204 (1:100; number KT022; Trans Genic Inc., Fukuoka, Japan) at 4°C overnight. ESCC tissues were incubated with rabbit antibody against CCL1 (1:50; number HPA049861; Atlas Antibody)/CCR8 (1:100; number NBP2-15768; Novus Biologicals), mouse antibody against CD204 (1:100; number KT022; Trans Genic Inc.), mouse antibody against forkhead box P3 (1:100; number 20034; Abcam), or sheep antibody against fibroblast activation protein (1:100; number AF3715; R&D systems) at 4°C overnight. TE-8, TE-9, and TE-15 cells were incubated with rabbit antibody against CCR8 (1:100; number ab32399; Abcam) at 4°C overnight. Cells that had been incubated with the primary antibodies were then incubated with Alexa Fluor 488–conjugated donkey anti-rabbit or sheep secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated donkey anti-mouse or rabbit secondary antibody (1:200; Jackson ImmunoReserch Laboratories) at room temperature for 1 hour. Nuclei were stained with DAPI (1:1000; number GV039; Wako). Images were obtained using the Zeiss LSM 700 laser-scanning microscope and analyzed using LSM ZEN 2009 (Carl Zeiss, Oberkochen, Germany). All in vitro experiments were performed in triplicate and were independently repeated three times. The results are expressed as means ± SEM. Significance was analyzed using two-sided t-tests and Tukey-Kramer tests for comparing more than two groups. The relationships between clinicopathologic factors and immunohistochemical results were estimated using the χ2 test. Kaplan-Meier curves were used to evaluate OS and disease-

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