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The S100A8–serum amyloid A3–TLR4 paracrine cascade establishes a pre-metastatic phase Sachie Hiratsuka1, Akira Watanabe2, Yoshiko Sakurai1, Sachiko Akashi-Takamura3, Sachie Ishibashi1, Kensuke Miyake3, Masabumi Shibuya4, Shizuo Akira5, Hiroyuki Aburatani2 and Yoshiro Maru1,6 A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs1,2 in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil3. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-κB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3–TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis. Although the seed-and-soil theory of metastasis was originally proposed more than a century ago4, the reason(s) behind the propensity for certain primary tumour cells to seed in particular organs is largely unknown. Upregulation of matrix metalloproteinase 9 (MMP9) in macrophages and endothelial cells, a process dependent on secretion of vascular endothelial growth factor (VEGF) from primary tumours, promotes tumour cell invasion5. Bone marrow-derived haematopoietic progenitor cells expressing VEGF receptor (VEGFR) 1 are known to form cellular clusters in pre-metastatic organs2. Our recent work has indicated that the inflammatory chemoattractants S100A8 and S100A9, whose expression in the pre-metastatic lung is induced by distant primary tumours, attract Mac 1+-myeloid cells3. As lung conditioned medium (LCM) stimulated with S100A8 is a better stimulant for the
migration of Mac 1+-myeloid and tumour cells than S100A8 alone, we hypothesize that paracrine stimulation of mediator(s) by S100A8 is responsible for this action. A member of the SAA family, SAA3 functions as a chemotactic agonist for phagocytes6–8 and was one of the top upregulated genes in pre-metastatic lungs in our previous microarray analyses3. We performed time-course experiments on SAA3 induction by primary tumours in several organs, both at the mRNA and protein levels, and found that its level of expression was higher in lungs than in liver or kidney (Fig. 1a). The pattern of induction was similar to that of S100A8 and S100A9. To identify cells producing SAA3 in response to primary tumours, we assessed SAA3 expression levels in endothelial cells and Mac 1+-myeloid cells of mice with Lewis lung carcinoma (LLC) and found that it was upregulated in both cell types in these mice (Supplementary Information, Fig. S1a). In immunohistochemistry experiments, faint signals of SAA3 were detected in Mac 1+-myeloid cells but not in VE-cadherin+-endothelial cells from normal mice (Supplementary Information, Fig. S1b). However, we observed strong expression of SAA3 in both cell types from tumour-bearing wild-type mice, (Supplementary Information, Fig. S1b). To test whether S100A8 regulates the expression of SAA3, we cultured lungs and livers in the presence of S100A8 protein, and measured the SAA3 mRNA level. S100A8 induced expression of SAA3 specifically in the lung but not in the liver (Fig. 1b). We also found that both S100A8 and lipopolysaccharide (LPS) stimulated SAA3 protein secretion into LCM. A neutralizing anti-S100A8 antibody3 inhibited S100A8-induced SAA3 protein secretion (Fig. 1c; Supplementary Information, Fig. S7). Induction of SAA3 by S100A8 was further confirmed in endothelial cells and alveolar macrophages purified from lungs (Fig. 1d; Supplementary Information, Fig. S7). Next, we directly stimulated the SAA3 promoter with S100A8 in a promoter-luciferase assay. We found an increase in SAA3 promoter activity of 1.7- to 4.0-fold in macrophages, mononuclear cells from bone marrow and MCF7 cells (Fig. 1e).
1 Department of Pharmacology, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. 2Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. 3Division of Infectious Genetics, Department of Microbiology and immunology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 4 Division of Genetics, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 5Department of Host Defense, Research Institute for Microbial Disease, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-1871, Japan. 6 Correspondence should be addressed to Y. M. (e-mail:
[email protected])
Received 28 July 2008; accepted 10 September 2008; published online 28 September 2008; DOI: 10.1038/ncb1794
nature cell biology volume 10 | number 11 | NOVEMBER 2008 © 2008 Macmillan Publishers Limited. All rights reserved.
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Figure 1 Effects of S100A8 and S100A9 on SAA3 gene expression and protein secretion. (a) RT–PCR (upper) and western blot analyses (lower) of S100A8, S100A9 and SAA3 in organs from normal (N) and tumour (L: LLC, B: B16)bearing mice 10 or 14 days after implantation. (b) RT–PCR analyses of SAA3 in lung and liver cultured with S100A8–GST (upper) and S100A8–FLAG (lower) proteins for 30 and 90 min. (pFLAG: control FLAG peptide). (c) Western blot analyses of SAA3 in conditioned medium. Lungs were incubated with mock (M, medium only), GST, A8–GST, A8–FLAG, pFLAG or LPS (L) in the presence
or absence of anti-S100A8 antibody (A8-Ab). (d) RT–PCR analysis of SAA3 in the lung endothelial cells (EC) and alveolar macrophages cultured with S100A8–GST. (e) Effects of S100A8–GST or GST on the luciferase activity of the SAA3 promoter in macrophages, mononuclear (MN) cells or MCF7 cells (*P
