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Pre-Metastatic Niche Formation Has Taken Its TOLL

2016; Cell Press; Volume: 30; Issue: 2 Linguagem: Inglês

10.1016/j.ccell.2016.07.012

1878-3686

Candia M. Kenific, Laura Nogués, David Lyden,

Cancer-related molecular mechanisms research

Pre-Metastatic Niches (PMNs) result from communications between primary tumors and the microenvironment of future distant metastasis via tumor-derived factors. In this issue of Cancer Cell, Liu et al. show that TLR3 activation in lung epithelial cells by tumor exosomal RNAs triggers neutrophil recruitment, which contributes to PMN formation and metastasis. Pre-Metastatic Niches (PMNs) result from communications between primary tumors and the microenvironment of future distant metastasis via tumor-derived factors. In this issue of Cancer Cell, Liu et al. show that TLR3 activation in lung epithelial cells by tumor exosomal RNAs triggers neutrophil recruitment, which contributes to PMN formation and metastasis. Metastasis, the process by which primary tumor cells colonize distant organs, is the major cause of cancer mortality. Cultivation of a hospitable Pre-Metastatic Niche (PMN) is well recognized as being essential for tumor cell growth at foreign sites. The initial discovery of the PMN revealed that tumor-secreted factors alter the host microenvironment in secondary organs by increasing fibronectin expression and promoting recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing bone-marrow-derived cells (BMDCs) (Kaplan et al., 2005Kaplan R.N. Riba R.D. Zacharoulis S. Bramley A.H. Vincent L. Costa C. MacDonald D.D. Jin D.K. Shido K. Kerns S.A. et al.Nature. 2005; 438: 820-827Crossref PubMed Scopus (2473) Google Scholar). These BMDCs—which also express other markers, such as VLA-4, Id3, and MMP9, required for homing to pre-metastatic sites—in turn produce cytokines that enable metastatic formation. Additionally, upregulation of S100A8 and S100A9 in the PMN leads to infiltration of myeloid cells that further support PMN formation (Hiratsuka et al., 2006Hiratsuka S. Watanabe A. Aburatani H. Maru Y. Nat. Cell Biol. 2006; 8: 1369-1375Crossref PubMed Scopus (820) Google Scholar). Among tumor-secreted factors, exosomes are critical for PMN establishment. They directly reprogram BMDCs to mobilize them to pre-metastatic sites (Peinado et al., 2012Peinado H. Alečković M. Lavotshkin S. Matei I. Costa-Silva B. Moreno-Bueno G. Hergueta-Redondo M. Williams C. García-Santos G. Ghajar C. et al.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2635) Google Scholar). They also target future metastatic sites to induce vascular leakiness and educate stromal cells (Costa-Silva et al., 2015Costa-Silva B. Aiello N.M. Ocean A.J. Singh S. Zhang H. Thakur B.K. Becker A. Hoshino A. Mark M.T. Molina H. et al.Nat. Cell Biol. 2015; 17: 816-826Crossref PubMed Scopus (1672) Google Scholar, Hoshino et al., 2015Hoshino A. Costa-Silva B. Shen T.L. Rodrigues G. Hashimoto A. Tesic Mark M. Molina H. Kohsaka S. Di Giannatale A. Ceder S. et al.Nature. 2015; 527: 329-335Crossref PubMed Scopus (2928) Google Scholar, Peinado et al., 2012Peinado H. Alečković M. Lavotshkin S. Matei I. Costa-Silva B. Moreno-Bueno G. Hergueta-Redondo M. Williams C. García-Santos G. Ghajar C. et al.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2635) Google Scholar). Stromal cell education further reinforces PMN formation by creating a pro-inflammatory microenvironment through recruitment of additional BMDCs. Importantly, selective packaging of exosomal cargoes, such as integrins, contributes to exosome-mediated organotropism (Hoshino et al., 2015Hoshino A. Costa-Silva B. Shen T.L. Rodrigues G. Hashimoto A. Tesic Mark M. Molina H. Kohsaka S. Di Giannatale A. Ceder S. et al.Nature. 2015; 527: 329-335Crossref PubMed Scopus (2928) Google Scholar). These observations illustrate the complex, organ-specific responses to tumor-secreted factors for PMN formation, pointing to the need for a comprehensive understanding of these pathways in different metastatic sites. In this issue of Cancer Cell, Liu et al. identify a previously unrecognized pathway through which TLR3 expressed by alveolar type II (AT-II) epithelial cells promotes lung PMN formation (Liu et al., 2016Liu Y. Gu Y. Han Y. Zhang Q. Jiang Z. Zhang X. Huang B. Xu X. Zheng J. Cao X. Cancer Cell. 2016; 30 (this issue): 243-256Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Previous work uncovered that TLR2 activation on host myeloid cells by tumor-derived cytokines contributes to formation of a permissive metastatic niche (Kim et al., 2009Kim S. Takahashi H. Lin W.W. Descargues P. Grivennikov S. Kim Y. Luo J.L. Karin M. Nature. 2009; 457: 102-106Crossref PubMed Scopus (889) Google Scholar). To better define the function of TLRs in cancer progression, Liu et al. first investigated the roles of TLR3, TLR4, and TLR9 in pulmonary metastasis. Strikingly, they found that growth of lung metastases was attenuated in Tlr3−/− mice but not in mice lacking Tlr4 or Tlr9. Primary tumor growth was not altered by TLR3 deficiency, suggesting that it specifically affects metastasis. Characterization of the PMN revealed that, compared to wild-type mice, pre-metastatic lungs of Tlr3−/− mice expressed low levels of the PMN markers Bv8, S100a8, S100a9, and MMP9 and exhibited reduced fibronectin deposition and BMDC recruitment, suggesting that TLR3 supports PMN establishment. Further assessment of PMN formation showed that Tlr3−/− mice exhibited a robust reduction in tumor-mediated neutrophil recruitment to lungs and that anti-Ly6G-mediated neutrophil depletion in wild-type mice abrogated lung metastasis, verifying a pro-metastatic role for neutrophils in the PMN. These results build on previous work showing that tumor-mediated neutrophil chemoattraction to pre-metastatic sites promotes metastatic colonization through multiple mechanisms, including selective activation of metastasis-initiating cells (Coffelt et al., 2016Coffelt S.B. Wellenstein M.D. de Visser K.E. Nat. Rev. Cancer. 2016; 16: 431-446Crossref PubMed Scopus (956) Google Scholar, Wculek and Malanchi, 2015Wculek S.K. Malanchi I. Nature. 2015; 528: 413-417Crossref PubMed Scopus (648) Google Scholar). However, neutrophils also inhibit metastasis (Coffelt et al., 2016Coffelt S.B. Wellenstein M.D. de Visser K.E. Nat. Rev. Cancer. 2016; 16: 431-446Crossref PubMed Scopus (956) Google Scholar). Furthermore, the difference between neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), also defined as CD11b+Ly6G+Ly6Clow/med, is currently unclear (Coffelt et al., 2016Coffelt S.B. Wellenstein M.D. de Visser K.E. Nat. Rev. Cancer. 2016; 16: 431-446Crossref PubMed Scopus (956) Google Scholar). The finding by Liu et al. that recruited neutrophils are also cKit+ suggests that they are most likely immature or precursor neutrophils, rather than mature ones (Coffelt et al., 2016Coffelt S.B. Wellenstein M.D. de Visser K.E. Nat. Rev. Cancer. 2016; 16: 431-446Crossref PubMed Scopus (956) Google Scholar). In light of these discrepancies, further study on the specific identity and function of neutrophils in mediating TLR3-dependent metastasis is necessary. It is also notable that recruitment of additional BMDCs to pre-metastatic sites is impaired in Tlr3−/− mice. Thus, according to the authors’ model (Figure 1), neutrophils may help orchestrate diverse immune cell responses. Although systemic knockout of Tlr3 significantly reduced PMN establishment, neutrophil recruitment, and metastasis, the cells in which TLR3 signaling was critical remained unknown. Gene expression and immunofluorescence experiments revealed that TLR3 is predominantly expressed by AT-II cells in pre-metastatic lungs (Liu et al., 2016Liu Y. Gu Y. Han Y. Zhang Q. Jiang Z. Zhang X. Huang B. Xu X. Zheng J. Cao X. Cancer Cell. 2016; 30 (this issue): 243-256Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Lung epithelial cell-specific re-expression of TLR3 via delivery of TLR-expressing recombinant adenovirus to the respiratory tract restored the metastatic potential of tumor cells in Tlr3−/− mice, indicating that AT-II cell TLR3 is essential for PMN formation and metastasis. TLR3 was also shown to regulate expression of cytokines by AT-II cells, suggesting that these lung stromal cells contribute to neutrophil recruitment. Analysis of exosome uptake corroborated previous work showing that AT-II cells are among the main lung cell types uptaking exosomes (Hoshino et al., 2015Hoshino A. Costa-Silva B. Shen T.L. Rodrigues G. Hashimoto A. Tesic Mark M. Molina H. Kohsaka S. Di Giannatale A. Ceder S. et al.Nature. 2015; 527: 329-335Crossref PubMed Scopus (2928) Google Scholar), indicating that exosomes are a key tumor-derived messenger promoting TLR3 activation. Accordingly, Tlr3−/− mice exhibited diminished exosome-induced PMN formation, cytokine production, neutrophil recruitment, and metastasis. Although these findings identify TLR3 as a mediator of exosome-dependent PMN formation, Tlr3−/− mice still exhibited increases in multiple PMN markers and neutrophil infiltration, indicating that exosomes activate these pathways through additional TLR3-independent mechanisms. Because Liu et al. identified neutrophils and monocytes as VEGFR1+ BMDCs that are recruited to pre-metastatic lungs, and because VEGFR1+ BMDCs can be educated by tumor exosomes (Peinado et al., 2012Peinado H. Alečković M. Lavotshkin S. Matei I. Costa-Silva B. Moreno-Bueno G. Hergueta-Redondo M. Williams C. García-Santos G. Ghajar C. et al.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2635) Google Scholar), such a TLR3-independent pathway may involve direct reprogramming of neutrophils by exosomes. TLR3 is known to detect double-stranded RNA (dsRNA) (Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4924) Google Scholar), and exosomes transfer RNA (Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar). Thus, the authors hypothesized that exosomal RNAs (exoRNAs) are cargoes that activate TLR3. Analysis of exoRNAs showed that the majority of exoRNAs consisted of non-coding RNA and that, within this group, small nuclear RNA (snRNA) was highly abundant. U1 snRNAs were particularly enriched; this was in stark contrast to tumor RNA, which consisted of 1,000-fold less U1 snRNA. The authors highlighted that the known secondary stem-loop structure of U1 snRNA forms double strands and therefore may also activate TLR3. Unlike work that has largely focused on regulation of metastasis by exosomal microRNAs (Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar), this result points to a previously unrecognized role for snRNAs in metastasis and suggests that, in addition to directly modulating gene expression in recipient cells, exosomal non-coding RNAs function via post-translational mechanisms. It remains to be determined whether snRNAs are functionally required for PMN formation in vivo, and the mechanisms regulating their selective packaging and enrichment in exosomes warrant further investigation. Although only the role of exoRNAs in promoting TLR3 activation in vitro was investigated, it appeared that TLR3 is likely upregulated by other exosomal cargoes, such as proteins or DNA. This finding illustrates the need to understand how multiple exosomal cargoes cooperate to elicit metastasis-promoting cellular responses. This study points to TLR3 as a viable therapeutic target against PMN formation and metastasis. Importantly, because PMN formation is associated with fibrosis and vascular leakiness, developing treatments directly targeting the PMN would be beneficial. Such therapy would be ideal in clinical cases in which metastases are not immediately evident but are likely to occur, or in which PMNs co-exist with metastatic tumors. These unmet clinical needs highlight the attractiveness of targeting TLR3, as well as other inducers of PMN formation. Although Liu et al. correlated high lung TLR3 expression with neutrophil infiltration and poor prognosis in lung cancer patients, these data may not accurately reflect the pathology of lung PMN formation induced by primary tumors originating elsewhere. Future efforts must focus better on understanding PMN formation in patients for confident identification of targeted therapies that, combined with treatments against primary tumor and disseminated tumor cells, may lead to successful eradication of metastasis. Tumor Exosomal RNAs Promote Lung Pre-metastatic Niche Formation by Activating Alveolar Epithelial TLR3 to Recruit NeutrophilsLiu et al.Cancer CellAugust 08, 2016In BriefLiu et al. demonstrate that TLR3 in host alveolar epithelial cells is critical for neutrophil recruitment and lung pre-metastatic niche formation. Mechanistically, small nuclear RNAs from primary tumor-derived exosomes activate TLR3, which leads to chemokine secretion and neutrophil infiltration. Full-Text PDF Open Archive