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A Link Between Inflammation and Metastasis

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A Link Between Inflammation and Metastasis Oncogene (2015) 34, 424–435 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4 MT Hansen1,8, B Forst1,8, N Cremers2,3, L Quagliata2, N Ambartsumian1,4, B Grum-Schwensen1, J Klingelho¨ fer1,4, A Abdul-Al1, P Herrmann5, M Osterland5, U Stein5, GH Nielsen6, PE Scherer7, E Lukanidin1, JP Sleeman2,3,9 and M Grigorian1,4,9 S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4- dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-kB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression. Oncogene (2015) 34, 424–435; doi:10.1038/onc.2013.568; published online 27 January 2014 Keywords: inflammation; metastasis; S100A4; serum amyloid A INTRODUCTION (MMPs).6–12 SAA can signal via Toll-like receptors (TLRs) and 9,13 The microenvironment surrounding tumor cells, including cells of nuclear factor-kB (NF-kB). In mice, SAA3 contributes to the 14 the immune system and proinflammatory factors, have a key role establishment of lung premetastatic niches, which are 15 in regulating metastasis formation.1,2 Although different microenvironments that foster metastasis formation. subclasses of immune cells can inhibit or promote metastasis, We have previously implicated the S100 family member S100A4 chronic inflammation in tumors generally predicts poor in both tumor progression and in inflammatory diseases such as 16–18 prognosis.3 Serum amyloid A (SAA) is a family of highly rheumatoid arthritis, psoriasis and dermato/polymyosites. homologous acute-phase proteins whose expression and S100 proteins require calcium-induced oligomerization for their accumulation in the blood is observed during inflammation and activity, and are involved in the regulation of proliferation, 19,20 has been associated with tumor progression and reduced survival survival, differentiation and motility. S100A4 expression is in many human cancers.4 SAA1 and SAA2 are the major acute- enhanced in highly metastatic CSML100 mouse mammary phase plasma isoforms. A third acute-phase isoform SAA3 is adenocarcinoma cells compared with poorly metastatic CSML0 expressed in rodents, but is a transcribed pseudogene in humans.5 cells, and functionally contributes to metastasis.21–25 Furthermore, In vivo concentrations of SAAs increase in the blood during the the metastatic behavior of the mouse adenocarcinoma cell line response to trauma, infection, inflammation and neoplasia, and VMR is determined by the stroma, and is stimulated by S100A4- serve to regulate homeostatically lipid metabolism and transport, expressing fibroblasts, indicating that S100A4 can promote immune cell chemotaxis and other inflammatory processes. SAA metastasis formation through both autocrine and paracrine proteins regulate the expression of cytokines including mechanisms.26 Clinical studies also demonstrate that augmented interleukins, granulocyte-colony-stimulating factor (G-CSF) and expression of S100A4 in primary tumors correlates with poor tumor necrosis factor-a, as well as matrix metalloproteinases prognosis (reviewed in Helfman et al.27). S100A4-mediated 1Danish Cancer Society Research Center, Copenhagen, Denmark; 2Universita¨tsmedizin Mannheim, University of Heidelberg, Mannheim, Germany; 3KIT Karlsruhe, Eggenstein- Leopoldshafen, Germany; 4Neuro-Oncology Group, Laboratory of Neural Plasticity, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, Copenhagen, Denmark; 5Experimental and Clinical Research Center, Charite´ Universita¨tsmedizin Berlin, at the Max-Delbru¨ck Center for Molecular Medicine, Berlin, Germany; 6Air Liquide Danmark A/S, Horsens, Denmark and 7Touchstone Diabetes Center, University of Texas Southwestern Medical Center, Dallas, TX, USA. Correspondence: Dr M Grigorian, Neuro-Oncology Group, Laboratory of Neural Plasticity, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, Blegdamsvej 3B, Copenhagen 2200, Denmark. E-mail: [email protected] 8These authors contributed equally to this work. 9These authors contributed equally to this work. Received 3 July 2013; revised 6 December 2013; accepted 7 December 2013; published online 27 January 2014 S100A4-induced SAA expression promotes metastasis MT Hansen et al 425 metastasis is associated with extensive T-cell infiltration into both expression by mouse recS100A4 (Figure 1e and Supplementary the primary tumor and sites of metastasis.28 Despite the extensive Figure S1D). In addition, mutant forms of hS100A4 (moA4mt1 and evidence implicating S100A4 in metastasis formation, the moA4mt2) that cannot form oligomers had an attenuated ability mechanism by which it exerts its effects remains uncertain. to induce SAA activation (Figure 1e and Supplementary Figure Here we found that SAA proteins are transcriptional targets of S1D). These data demonstrate that S100A4 specifically stimulates S100A4. In turn, SAA proteins stimulated both their own the expression of SAA in VMR cells. transcription and that of a variety of cytokines and MMPs, and promoted chemotactic migration and adhesion to fibronectin by tumor cells. Ectopic SAA expression in experimental tumors SAA augments the metastasis-associated properties of tumor cells sufficed to augment strongly metastasis formation, and was in vitro accompanied by a marked immune cell infiltration. Consistently, We next examined whether SAA proteins modify tumor cell coexpression of SAA and S100A4 correlated with poor prognosis properties associated with metastasis. VMR cells were transduced in human colorectal cancer patients. These data provide an with SAA1 and SAA3 retroviral expression vectors to obtain the important link between the metastasis-promoting and proinflam- stable SAA-expressing cell lines VMR/SAA1 and VMR/SAA3. Cells matory effects of S100A4, and suggest that the induction of SAA transduced with the empty vector (VMR/CTL) served as controls. expression is a major means by which S100A4 exerts these effects. Expression and secretion of SAA proteins was verified by western blotting (Supplementary Figure S2A). In subsequent experiments, the use of retrovirally transduced cells compared with exo- RESULTS genously added SAA protein (either as CM or as recombinant S100A4 induces expression of proinflammatory genes, including protein) allowed paracrine and autocrine effects to be compared. SAA family members Both VMR/SAA1 and VMR/SAA3 cells adhered much more To investigate how S100A4 promotes metastasis, we used strongly to fibronectin than the VMR/CTL cells (Figure 2a), but not microarrays to transcriptionally profile VMR tumor cells treated or to laminin or collagen (Supplementary Figure S2B). Effects of SAA non-treated for 24 h with the active multimeric form of the on cell motility were assessed by incubating wounded monolayers recombinant human S100A4 (hS100A4) protein. Of the 75 genes of mouse CSML100 and human MDA-MB-231 and SW480 tumor upregulated in response to hS100A4, more than 30% are cells with CM from VMR/SAA1, VMR/SAA3 and VMR/CTL cells, or associated with inflammation (Table 1). The most strongly with medium supplemented with recSAAs. Effects on the motility upregulated genes included the acute-phase reactants (SAA1 and of mouse embryonic fibroblasts were also examined, as S100A4 SAA3), cytokines and their receptors (CXCL1, CSF1, CCL4, INFAR2), stimulates the motility of activated fibroblasts and recruits them to the proinflammatory S100 family member S100A8, inflammation- tumors, promoting metastasis.29 In all cases, SAA-containing associated proteases and immune cell-specific genes. media induced a significant increase in motility compared with Given their upregulation in response to S100A4 and the control media (Figures 2b and c and Supplementary Figures S2C– literature correlating their expression with poor prognosis, we E). Both recSAA1 and recSAA3 also exerted chemotactic effects in determined whether SAA1 and SAA3 (hereafter referred to a dose-dependent manner on CSML100 tumor cells after 6 h in generically as SAA) might be mediators of S100A4-induced Transwell migration/invasion assays (Figure 2d). Significantly
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