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

doi:10.1007/s11515-012-1237-8

Front Biol

2012, Vol. 7

Issue (4) : 359-367 https://doi.org/10.1007/s11515-012-1237-8

RESEARCH ARTICLE

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

Yingbin GE^1,2, Rikka AZUMA³, Bethsebah GEKONGE⁴, Alfonso LOPEZ-CORAL⁵, Min XIAO¹, Gao ZHANG¹, Xiaowei XU⁶, Luis J. MONTANER⁴, Zhi WEI⁷, Meenhard HERLYN¹, Tao WANG¹(

), Russel E. KAUFMAN¹

1. Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, USA; 2. Department of Physiology, Nanjing Medical University, Nanjing 210029, China; 3. Undergraduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA; 4. Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA; 5. Graduate Program, The Catholic University of America, Washington DC 20064, USA; 6. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 7. Department of Computer Science, New Jersey Institute of Technology, NJ 07102, USA

Download: PDF(434 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

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

Corresponding Author(s): WANG Tao,Email:twang@wistar.org

Issue Date: 01 August 2012

Cite this article:

Yingbin GE,Alfonso LOPEZ-CORAL,Min XIAO, et al. Induction of metallothionein expression during monocyte to melanoma-associated macrophage differentiation[J]. Front Biol, 2012, 7(4): 359-367.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-012-1237-8
https://academic.hep.com.cn/fib/EN/Y2012/V7/I4/359

Fig.1 Induction of expression of metallothionein genes in M-CSF/Mф and MCIM-Mф. (A) Heatmap of gene expression of metallothionein genes in MCIM-Mф compared to monocytes. (B) Monocytes from healthy donors were incubated with melanoma-conditioned media for 7 days. Cells were harvested, total RNA isolated and real-time PCR was used to verify expression of MT genes in M-CSF/Mф and MCIM-Mф, including MT1G, MT1A, MT1E, MT1F and MT1H. Data are representative of 3 independent experiments with 3 healthy donors.

Fig.2 Expression of metallothionein in MCIM-Mф. (A) Expression of metallothionein in MCIM-Mф. FACS analysis was performed to quantify intracellular metallothionein expression in monocytes and MCIM-Mф. Gray shadow fills= isotype matched control; black lines= primary antibodies. (B) Monocytes and MCIM-Mф were fixed, cytospinned on glass slides, and stained with rabbit anti-metallothionein polyclonal antibody for immunofluorescence microscopy. Scale bar= 50 μm. (C) Formalin-fixed, paraffin embedded melanoma tissue sections were deparaffinized followed by antigen retrieval in citrate buffer. Melanoma tissues were then stained with anti-metallothionein antibody, and revealed that metallothionein is expressed in inflammatory cells, but not in tumor cells of primary melanoma lesion. Scale bar= 50 μm. Data are representative of 4 primary melanoma lesions.

Fig.3 Metallothionein is co-localized with CD68 and CD163 in melanoma tissues. Co-staining of CD68 (A) and CD163 (B) with metallothionein in human melanoma patient tissues. Melanoma tissues were treated as in Figure 3C. Anti- metallothionein and anti-CD68, CD163 antibodies were used to measure the co-localization of metallothionein with CD68 and CD163. Data are representative of 3 primary melanoma lesions.

Fig.4 Increased expression of intracellular zinc in MCIM-Mф. (A) DCF uptake assay was used to analyze the level of intracellular zinc in monocytes and MCIM-Mф. Gray= unstained control; black lines= DCF staining. (B) Real-time PCR was used to analyze the expression of zinc importer gene Zip8 in monocytes, M-CSF/Mф and MCIM-Mф.

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 pmid:10711641
2	Br?cker 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 doi: 10.1002/ijc.2910410415 pmid:3128489
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 doi: 10.1007/BF01069744 pmid:7519954
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 doi: 10.1039/b926662c pmid:21069178
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 pmid:2203773
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 doi: 10.1093/carcin/24.1.25 pmid:12538345
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 doi: 10.1182/blood-2007-02-072587 pmid:17848619
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 doi: 10.1038/sj.jid.5700827 pmid:17460736
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 doi: 10.1016/S0076-6879(02)53070-6 pmid:12078520
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 doi: 10.1016/j.jsb.2005.09.012 pmid:16495082
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 doi: 10.1158/1055-9965.EPI-04-0659 pmid:15894685
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 doi: 10.1002/ijc.11006 pmid:12640681
13	Jensen T O, Schmidt H, M?ller H J, H?yer 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 doi: 10.1200/JCO.2008.19.9919 pmid:19528371
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 doi: 10.1038/sj.onc.1208206 pmid:15735762
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 doi: 10.1101/gr.091447.109 pmid:19491193
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 doi: 10.1042/0264-6021:3550473 pmid:11284736
17	Lewis C E, Pollard J W (2006). Distinct role of macrophages in different tumor microenvironments. Cancer Res , 66(2): 605-612 doi: 10.1158/0008-5472.CAN-05-4005 pmid:16423985
18	M?kitie T, Summanen P, Tarkkanen A, Kivel? T (2001). Tumor-infiltrating macrophages (CD68⁺ cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci , 42(7): 1414-1421 pmid:11381040
19	Mantovani A, Sica A (2010). Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol , 22(2): 231-237 doi: 10.1016/j.coi.2010.01.009 pmid:20144856
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 pmid:10096565
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 doi: 10.1084/jem.190.2.293 pmid:10432291
22	Pernick N L, DaSilva M, Gangi M D, Crissman J, Adsay V (1999). “Histiocytic markers” in melanoma. Mod Pathol , 12(11): 1072-1077 pmid:10574605
23	Pollard J W (2004). Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer , 4(1): 71-78 doi: 10.1038/nrc1256 pmid:14708027
24	Qian B Z, Pollard J W (2010). Macrophage diversity enhances tumor progression and metastasis. Cell , 141(1): 39-51 doi: 10.1016/j.cell.2010.03.014 pmid:20371344
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 pmid:10741707
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 doi: 10.1016/S0360-3016(98)00329-0 pmid:9845085
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 doi: 10.1189/jlb.0110051 pmid:20551211
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 doi: 10.1074/jbc.M109.042671 pmid:19833726
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 doi: 10.1016/S0344-0338(97)80103-3 pmid:9342756
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 pmid:9701160
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 doi: 10.1517/14728222.11.9.1219 pmid:17845147
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 doi: 10.4049/jimmunol.1000413 pmid:20530259
33	Sugiura T, Kuroda E, Yamashita U (2004). Dysfunction of macrophages in metallothionein-knock out mice. J UOEH , 26(2): 193-205 pmid:15244072
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 doi: 10.1159/000218032 pmid:19420986
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 doi: 10.1097/00008390-200510000-00010 pmid:16179869
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 April12, 2012
37	Weinlich G (2009). Metallothionein-overexpression as a prognostic marker in melanoma. G Ital Dermatol Venereol , 144(1): 27-38 pmid:19218909
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 doi: 10.1046/j.1365-2133.2003.05472.x pmid:14510986
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 doi: 10.1038/sj.bjc.6603028 pmid:16508630
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 pmid:17447982
41	Weinlich G, Zelger B (2007). Metallothionein overexpression, a highly significant prognostic factor in thin melanoma. Histopathology , 51(2): 280-283 doi: 10.1111/j.1365-2559.2007.02744.x pmid:17593214
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 pmid:17914565
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 doi: 10.1083/jcb.200702081 pmid:17502426
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 doi: 10.1038/nature09666 pmid:21248750

Viewed

Full text

Abstract

Cited

Shared

Discussed