Induction of metallothionein expression during monocyte to melanoma-associated macrophage differentiation

Abstract

Tumor-associated macrophages (TAMs) play a critical role in melanoma growth and metastasis. Infiltration of TAMs correlates with the poor prognosis of melanoma. TAMs are differentiated from monocytes in response to the tumor microenvironment cue. However, the mechanism how TAMs adapt to the tumor microenvironment after differentiation from monocytes is not fully understood. In addition, specific identification of TAMs in melanoma is difficult because the expression of the most commonly used macrophage marker, CD68, is also expressed in melanoma cells. In an earlier study, we found by gene microarray analysis that seven members of the metallothionein (MTs) family were upregulated in melanoma-conditioned medium induced macrophages (MCIM-Mф). MTs have been implicated in zinc metabolism and inflammation. In the present study, we confirmed that expression of metallothionein is induced in M-CSF differentiated macrophages (M-CSF/Mф) and MCIM-Mф at both the mRNA and protein levels using real-time PCR, immunofluorescence, and western blot analysis. Furthermore, we demonstrated the presence of metallothionein in melanoma tissues in vivo and that metallothionein was co-localized with TAMs markers, CD68 and CD163. Finally, we demonstrated the induction of the zinc importer gene Zip8 both in M-CSF/Mф and MCIM-Mф. Our study identifies metallothionein as a novel marker for TAMs and suggests that metallothionein might play important roles in macrophage adaptation and function in the tumor microenvironment.

Keywords

melanoma / macrophages / metallothionein

References

[1] Bernengo M G, Quaglino P, Cappello N, Lisa F, Osella-Abate S, Fierro M T (2000). Macrophage-mediated immunostimulation modulates therapeutic efficacy of interleukin-2 based chemoimmunotherapy in advanced metastatic melanoma patients. Melanoma Res, 10(1): 55-65

[2] Bröcker E B, Zwadlo G, Holzmann B, Macher E, Sorg C (1988). Inflammatory cell infiltrates in human melanoma at different stages of tumor progression. Int J Cancer, 41(4): 562-567

[3] Cassidy M, Loftus B, Whelan A, Sabt B, Hickey D, Henry K, Leader M (1994). KP-1: not a specific marker. Staining of 137 sarcomas, 48 lymphomas, 28 carcinomas, 7 malignant melanomas and 8 cystosarcoma phyllodes. Virchows Arch, 424(6): 635-640

[4] Colvin R A, Holmes W R, Fontaine C P, Maret W (2010). Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics, 2(5): 306-317

[5] De S K, McMaster M T, Andrews G K (1990). Endotoxin induction of murine metallothionein gene expression. J Biol Chem, 265(25): 15267-15274

[6] Deng D, El-Rifai W, Ji J, Zhu B, Trampont P, Li J, Smith M F, Powel S M (2003). Hypermethylation of metallothionein-3 CpG island in gastric carcinoma. Carcinogenesis, 24(1): 25-29

[7] Duluc D, Delneste Y, Tan F, Moles M P, Grimaud L, Lenoir J, Preisser L, Anegon I, Catala L, Ifrah N, Descamps P, Gamelin E, Gascan H, Hebbar M, Jeannin P (2007). Tumor-associated leukemia inhibitory factor and IL-6 skew monocyte differentiation into tumor-associated macrophage-like cells. Blood, 110(13): 4319-4330

[8] Gazzaniga S, Bravo A I, Guglielmotti A, van Rooijen N, Maschi F, Vecchi A, Mantovani A, Mordoh J, Wainstok R (2007). Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft. J Invest Dermatol, 127(8): 2031-2041

[9] Ghoshal K, Majumder S, Jacob S T (2002). Analysis of promoter methylation and its role in silencing metallothionein I gene expression in tumor cells. Methods Enzymol, 353: 476-486

[10] Glesne D, Vogt S, Maser J, Legnini D, Huberman E (2006). Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells. J Struct Biol, 155(1): 2-11

[11] Henrique R, Jerónimo C, Hoque M O, Nomoto S, Carvalho A L, Costa V L, Oliveira J, Teixeira M R, Lopes C, Sidransky D (2005). MT1G hypermethylation is associated with higher tumor stage in prostate cancer. Cancer Epidemiol Biomarkers Prev, 14(5): 1274-1278

[12] Huang Y, de la Chapelle A, Pellegata N S (2003). Hypermethylation, but not LOH, is associated with the low expression of MT1G and CRABP1 in papillary thyroid carcinoma. Int J Cancer, 104(6): 735-744

[13] Jensen T O, Schmidt H, Møller H J, Høyer M, Maniecki M B, Sjoegren P, Christensen I J, Steiniche T (2009). Macrophage markers in serum and tumor have prognostic impact in American Joint Committee on Cancer stage I/II melanoma. J Clin Oncol, 27(20): 3330-3337

[14] Joshi B, Ordonez-Ercan D, Dasgupta P, Chellappan S (2005). Induction of human metallothionein 1G promoter by VEGF and heavy metals: differential involvement of E2F and metal transcription factors. Oncogene, 24(13): 2204-2217

[15] Koga Y, Pelizzola M, Cheng E, Krauthammer M, Sznol M, Ariyan S, Narayan D, Molinaro A M, Halaban R, Weissman S M (2009). Genome-wide screen of promoter methylation identifies novel markers in melanoma. Genome Res, 19(8): 1462-1470

[16] Levadoux-Martin M, Hesketh J E, Beattie J H, Wallace H M (2001). Influence of metallothionein-1 localization on its function. Biochem J, 355(Pt 2): 473-479

[17] Lewis C E, Pollard J W (2006). Distinct role of macrophages in different tumor microenvironments. Cancer Res, 66(2): 605-612

[18] Mäkitie T, Summanen P, Tarkkanen A, Kivelä T (2001). Tumor-infiltrating macrophages (CD68⁺ cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci, 42(7): 1414-1421

[19] Mantovani A, Sica A (2010). Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol, 22(2): 231-237

[20] Murphy B J, Andrews G K, Bittel D, Discher D J, McCue J, Green C J, Yanovsky M, Giaccia A, Sutherland R M, Laderoute K R, Webster K A (1999). Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res, 59(6): 1315-1322

[21] Niida S, Kaku M, Amano H, Yoshida H, Kataoka H, Nishikawa S, Tanne K, Maeda N, Nishikawa S, Kodama H (1999). Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med, 190(2): 293-298

[22] Pernick N L, DaSilva M, Gangi M D, Crissman J, Adsay V (1999). “Histiocytic markers” in melanoma. Mod Pathol, 12(11): 1072-1077

[23] Pollard J W (2004). Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer, 4(1): 71-78

[24] Qian B Z, Pollard J W (2010). Macrophage diversity enhances tumor progression and metastasis. Cell, 141(1): 39-51

[25] Raleigh J A, Chou S C, Calkins-Adams D P, Ballenger C A, Novotny D B, Varia M A (2000). A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. Clin Cancer Res, 6(3): 855-862

[26] Raleigh J A, Chou S C, Tables L, Suchindran S, Varia M A, Horsman M R (1998). Relationship of hypoxia to metallothionein expression in murine tumors. Int J Radiat Oncol Biol Phys, 42(4): 727-730

[27] Raymond A D, Gekonge B, Giri M S, Hancock A, Papasavvas E, Chehimi J, Kossenkov A V, Nicols C, Yousef M, Mounzer K, Shull J, Kostman J, Showe L, Montaner L J (2010). Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia. J Leukoc Biol, 88(3): 589-596

[28] Roca H, Varsos Z S, Sud S, Craig M J, Ying C, Pienta K J (2009). CCL2 and interleukin-6 promote survival of human CD11b⁺ peripheral blood mononuclear cells and induce M2-type macrophage polarization. J Biol Chem, 284(49): 34342-34354

[29] Shah I A, Gani O S, Wheler L (1997). Comparative immunoreactivity of CD-68 and HMB-45 in malignant melanoma, neural tumors and nevi. Pathol Res Pract, 193(7): 497-502

[30] Shankar A H, Prasad A S (1998). Zinc and immune function: the biological basis of altered resistance to infection. Am J Clin Nutr, 68(2 Suppl): 447S-463S

[31] Sica A, Rubino L, Mancino A, Larghi P, Porta C, Rimoldi M, Solinas G, Locati M, Allavena P, Mantovani A (2007). Targeting tumour-associated macrophages. Expert Opin Ther Targets, 11(9): 1219-1229

[32] Solinas G, Schiarea S, Liguori M, Fabbri M, Pesce S, Zammataro L, Pasqualini F, Nebuloni M, Chiabrando C, Mantovani A, Allavena P (2010). Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility. J Immunol, 185(1): 642-652

[33] Sugiura T, Kuroda E, Yamashita U (2004). Dysfunction of macrophages in metallothionein-knock out mice. J UOEH, 26(2): 193-205

[34] Tse K Y, Liu V W, Chan D W, Chiu P M, Tam K F, Chan K K, Liao X Y, Cheung A N, Ngan H Y (2009). Epigenetic alteration of the metallothionein 1E gene in human endometrial carcinomas. Tumour Biol, 30(2): 93-99

[35] Varney M L, Johansson S L, Singh R K (2005). Tumour-associated macrophage infiltration, neovascularization and aggressiveness in malignant melanoma: role of monocyte chemotactic protein-1 and vascular endothelial growth factor-A. Melanoma Res, 15(5): 417-425

[36] Wang T, Ge Y, Xiao M, Lopez-Coral A, Azuma R, Somasundaram R, Zhang G, Wei Z, Xu X, Rauscher Iii F J (2012). Melanoma-Derived Conditioned Media Efficiently Induce the Differentiation of Monocytes to Macrophages that Display a Highly Invasive Gene Signature. Pigment Cell Melanoma Res, Online Available <month>April</month><day>12</day>, 2012

[37] Weinlich G (2009). Metallothionein-overexpression as a prognostic marker in melanoma. G Ital Dermatol Venereol, 144(1): 27-38

[38] Weinlich G, Bitterlich W, Mayr V, Fritsch P O, Zelger B (2003). Metallothionein-overexpression as a prognostic factor for progression and survival in melanoma. A prospective study on 520 patients. Br J Dermatol, 149(3): 535-541

[39] Weinlich G, Eisendle K, Hassler E, Baltaci M, Fritsch P O, Zelger B (2006). Metallothionein- overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients. Br J Cancer, 94(6): 835-841

[40] Weinlich G, Topar G, Eisendle K, Fritsch P O, Zelger B (2007). Comparison of metallothionein-overexpression with sentinel lymph node biopsy as prognostic factors in melanoma. J Eur Acad Dermatol Venereol, 21(5): 669-677

[41] Weinlich G, Zelger B (2007). Metallothionein overexpression, a highly significant prognostic factor in thin melanoma. Histopathology, 51(2): 280-283

[42] Yamasaki M, Nomura T, Sato F, Mimata H (2007a). Metallothionein is up-regulated under hypoxia and promotes the survival of human prostate cancer cells. Oncol Rep, 18(5): 1145-1153

[43] Yamasaki S, Sakata-Sogawa K, Hasegawa A, Suzuki T, Kabu K, Sato E, Kurosaki T, Yamashita S, Tokunaga M, Nishida K, Hirano T (2007b). Zinc is a novel intracellular second messenger. J Cell Biol, 177(4): 637-645

[44] Zaidi M R, Davis S, Noonan F P, Graff-Cherry C, Hawley T S, Walker R L, Feigenbaum L, Fuchs E, Lyakh L, Young H A, Hornyak T J, Arnheiter H, Trinchieri G, Meltzer P S, De Fabo E C, Merlino G (2011). Interferon-γ links ultraviolet radiation to melanomagenesis in mice. Nature, 469(7331): 548-553

Acknowledgments

We thank the Wistar Institute Cancer Center Flow Cytometry Core Facility for helping with instrument setup and data analysis, the Microscopy Core Facility for imaging, and the Research Supply Center for cell culture supplies and reagents.

This work was supported by grants from the National Institutes of Health (5P30CA 010815-42) and the Commonwealth Universal Research Enhancement Program of the Pennsylvania Department of Health (R.E.K, L.J.M, M.H.), The Wistar Institute Intramural grants and the W.W. Smith Foundation for R.E.K and T.W., the National Institutes of Health grants for M.H. (CA047159, CA025874, CA114046), and the Philadelphia Foundation and Miller family grant for L.J.M.

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