Tumor Extracellular Vesicles Impede Interferon Alert Responses
Authors: Candia M. Kenific, Gang Wang, David Lyden
Journal: Cancer Cell (2019)

Tumor-derived extracellular vesicles promote metastasis by inducing functional changes in cells at pre-metastatic sites conducive for tumor cell colonization. In this issue of Cancer Cell, Ortiz and colleagues show that type I interferon regulates extracellular vesicle uptake and that modulating this pathway holds promise for treating metastasis. Tumor-derived extracellular vesicles promote metastasis by inducing functional changes in cells at pre-metastatic sites conducive for tumor cell colonization. In this issue of Cancer Cell, Ortiz and colleagues show that type I interferon regulates extracellular vesicle uptake and that modulating this pathway holds promise for treating metastasis. Key rate-limiting steps of metastasis, the main cause of cancer mortality, include growth of tumor cells in foreign, inhospitable microenvironments at distant organs. To overcome this obstacle, tumor cells induce formation of specialized sites, termed pre-metastatic niches (PMNs), conducive to their colonization at metastatic organs (Peinado et al., 2017Peinado H. Zhang H. Matei I.R. Costa-Silva B. Hoshino A. Rodrigues G. Psaila B. Kaplan R.N. Bromberg J.F. Kang Y. et al.Pre-metastatic niches: organ-specific homes for metastases.Nat. Rev. Cancer. 2017; 17: 302-317Crossref PubMed Scopus (936) Google Scholar). PMNs are established before tumor cell arrival and are characterized by several hallmarks, namely vascular leakiness, fibronectin deposition, and bone marrow-derived cell (BMDC) recruitment (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.VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.Nature. 2005; 438: 820-827Crossref PubMed Scopus (2467) 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.Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2622) Google Scholar). The essential role of PMNs in supporting metastasis has been established for multiple cancers, including melanoma and pancreatic and breast carcinomas (Peinado et al., 2017Peinado H. Zhang H. Matei I.R. Costa-Silva B. Hoshino A. Rodrigues G. Psaila B. Kaplan R.N. Bromberg J.F. Kang Y. et al.Pre-metastatic niches: organ-specific homes for metastases.Nat. Rev. Cancer. 2017; 17: 302-317Crossref PubMed Scopus (936) Google Scholar), underscoring the need to rigorously dissect mechanisms of PMN formation to identify therapies targeting this critical driver of metastasis. PMNs are induced by tumor-secreted factors, including extracellular vesicles (EVs). EVs are a heterogeneous population of vesicles released by cells through outward budding of the plasma membrane (microvesicles) or via secretion of small vesicles of endocytic origin (exosomes) (Becker et al., 2016Becker A. Thakur B.K. Weiss J.M. Kim H.S. Peinado H. Lyden D. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis.Cancer Cell. 2016; 30: 836-848Abstract Full Text Full Text PDF PubMed Scopus (1042) Google Scholar). EVs act as intercellular messengers carrying bioactive molecules, including nucleic acids, proteins, and lipids; upon uptake, these factors induce functional changes in recipient cells. For example, uptake of EVs harboring Met by BMDCs promotes homing to PMNs, whereas stromal residents, such as lung fibroblasts, lung epithelial cells, and hepatic Kupffer cells, rely on exosomal integrins, small RNAs, and macrophage migration inhibitory factor, respectively, to cultivate PMNs (Becker et al., 2016Becker A. Thakur B.K. Weiss J.M. Kim H.S. Peinado H. Lyden D. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis.Cancer Cell. 2016; 30: 836-848Abstract Full Text Full Text PDF PubMed Scopus (1042) 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.Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2622) Google Scholar, Peinado et al., 2017Peinado H. Zhang H. Matei I.R. Costa-Silva B. Hoshino A. Rodrigues G. Psaila B. Kaplan R.N. Bromberg J.F. Kang Y. et al.Pre-metastatic niches: organ-specific homes for metastases.Nat. Rev. Cancer. 2017; 17: 302-317Crossref PubMed Scopus (936) Google Scholar). Despite this knowledge, mechanisms regulating tumor EV (TEV) uptake remain poorly defined. In this issue of Cancer Cell, Ortiz et al. describe a role for type I interferon (IFN) signaling in PMN formation by modulating TEV uptake, and they harness this information to identify a small molecule inhibitor of PMNs (Ortiz et al., 2019Ortiz A. Gui J. Zahedi F. Yu P. Cho C. Bhattacharya S. Carbone C.J. Yu Q. Katlinski K.V. Katlinskaya Y.V. et al.An interferon-driven oxysterol-based defense against tumor-derived extracellular vesicles.Cancer Cell. 2019; 35 (this issue): 33-45Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To gain insight into TEV uptake, the authors screened a human melanoma cell line model for TEV-induced changes in cell surface receptor expression and identified IFNAR1, a type I IFN receptor subunit, as being downregulated. TEVs stimulated p38-mediated IFNAR1 phosphorylation and subsequent ubiquitination, resulting in IFNAR1 degradation. Importantly, melanoma patient (MP)-derived plasma EVs (MPEVs) decreased monocyte IFNAR1 expression in vitro, and MP peripheral blood cells had reduced IFNAR1 levels, exhibiting the clinical relevance of IFNAR1 degradation. Type I IFN signaling is the first line of antiviral defense (Schulz and Mossman, 2016Schulz K.S. Mossman K.L. Viral evasion strategies in type I IFN signaling - a summary of recent developments.Front. Immunol. 2016; 7: 498Crossref PubMed Scopus (88) Google Scholar). Upon viral infection, cells activate type I IFN, triggering expression of IFN-stimulated genes (ISGs); ISGs encode factors that interfere with viral infection to maintain an antiviral state. Viruses employ mechanisms, including IFNAR1 downregulation, to subvert this defense, illustrating similarities between mechanisms of TEVs and viruses. Accordingly, multiple lines of evidence demonstrating the functional importance of IFNAR1 downregulation for TEV uptake are provided. First, the authors confirmed TEV-mediated IFNAR1 degradation impairs ISG expression. Additionally, they demonstrated expression of degradation-resistant mutant IFNAR1 ("SA") allows sustained IFN signaling despite TEV exposure, decreasing further TEV uptake. Thus, similar to viral responses, cells engage type I IFN pathways upon TEV exposure. TEVs attenuate this defense by downregulating IFNAR1 allowing further TEV uptake, whereas persistent type I IFN activation inhibits continued TEV uptake. In agreement, the authors also demonstrated that normal fibroblast EVs (FEVs) induced a potent type I IFN signaling response, which correlated with their inability to suppress IFNAR1 and be continually taken up. These findings indicate cells employ mechanisms to prevent unwanted EV uptake and TEVs carry unique cargo that allows them to escape this host defense. Future work should investigate whether IFN-regulated pathways impact normal physiology and development and should define TEV-specific factors contributing to evasion of IFN defense. Using B16F10 mouse melanoma, the authors also showed inactivation of IFNAR1 is required for lung PMN formation and metastasis. Compared to wild-type mice, mice expressing SA IFNAR1 displayed impaired PMN formation when educated with B16F10 TEVs. Furthermore, spontaneous metastasis was reduced in SA mutant compared to wild-type B16F10 tumor-bearing mice, suggesting TEV-dependent IFN inhibition is required for metastasis formation. These results expand previous work showing IFN possesses anti-metastatic activity and identify TEV uptake inhibition as a potential mechanism for IFN-based treatment (Parker et al., 2016Parker B.S. Rautela J. Hertzog P.J. Antitumour actions of interferons: implications for cancer therapy.Nat. Rev. Cancer. 2016; 16: 131-144Crossref PubMed Scopus (532) Google Scholar). Of the numerous factors encoded by ISGs, expression of cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-hydroxysterol (25HC), has been shown to block fusion of viruses with cellular membranes (Liu et al., 2013Liu S.Y. Aliyari R. Chikere K. Li G. Marsden M.D. Smith J.K. Pernet O. Guo H. Nusbaum R. Zack J.A. et al.Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol.Immunity. 2013; 38: 92-105Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Thus, the authors explored whether this ISG can block EV uptake. Consistent with the ability of TEVs to inhibit IFN signaling, TEVs suppressed CH25H expression, whereas treatment of cells with 25HC decreased TEV uptake. Hence, similar to antiviral pathways, CH25H is an ISG that negatively regulates EV uptake, and TEVs promote their own uptake by downregulating type I IFN-dependent 25HC synthesis. As expected, CH25H inhibited PMN formation during TEV education in vivo and impaired metastasis in B16F10 tumor-bearing mice. Furthermore, MPEVs decreased CH25H expression in vitro, and CH25H expression was lowest in leukocytes from metastatic MPs compared to non-metastatic MP or healthy donor leukocytes, demonstrating the clinical relevance of CH25H. These findings reveal how TEVs thwart host defenses to promote uptake and condition PMNs, suggesting that exploiting anti-uptake pathways may be a strategy to target PMNs and metastasis. Therefore, the authors searched for small molecules that could mimic the action of 25HC. They identified the anti-hypertensive drug reserpine and showed it impaired TEV uptake and consequent PMN formation in vivo. Additionally, the authors modeled adjuvant/neoadjuvant reserpine therapy by administering the agent to mice before and after surgical excision of B16F10 tumors. They found that reserpine appeared to completely abrogate lung metastasis and improve overall survival of mice. Already approved for clinical use, reserpine is an attractive candidate that may be repurposed for treating metastatic melanoma. Overall, this study by Ortiz and colleagues builds upon prior PMN studies, advancing our understanding of TEV uptake by PMN cells (Figure 1). It also exposes IFN signaling as a natural defender against unwanted EV uptake during normal cellular homeostasis, illuminating striking parallels between viruses and EVs. Future studies should address how generalizable this uptake mechanism is to various normal cell types involved in PMN formation; type I IFN can be activated by many cell types, suggesting its broad importance (Parker et al., 2016Parker B.S. Rautela J. Hertzog P.J. Antitumour actions of interferons: implications for cancer therapy.Nat. Rev. Cancer. 2016; 16: 131-144Crossref PubMed Scopus (532) Google Scholar, Schulz and Mossman, 2016Schulz K.S. Mossman K.L. Viral evasion strategies in type I IFN signaling - a summary of recent developments.Front. Immunol. 2016; 7: 498Crossref PubMed Scopus (88) Google Scholar). Moreover, the screen for TEV-induced changes in cell surface receptors identified multiple candidates that should also be interrogated for their ability to fine-tune EV uptake. Though this study identified IFNAR1 as a negative regulator of uptake, positive mediators of TEV uptake are unknown and may serve as therapeutic targets. This study also expands upon recent work showing that ISGs are induced in tumor cells by stromal EVs in breast cancer (Nabet et al., 2017Nabet B.Y. Qiu Y. Shabason J.E. Wu T.J. Yoon T. Kim B.C. Benci J.L. DeMichele A.M. Tchou J. Marcotrigiano J. Minn A.J. Exosome RNA unshielding couples stromal activation to pattern recognition receptor signaling in cancer.Cell. 2017; 170: 352-366.e13Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). ISG expression was promoted by RIG-I-mediated intracellular sensing of stromal EV RNA, and ultimately it functioned to support tumor progression and metastasis. Taken together with the current study by Ortiz and colleagues, these results highlight that EVs elicit diverse IFN signaling-related responses via "viral mimicry" with varied outcomes on tumorigenesis and metastasis, underscoring the need for context-dependent characterization of therapies targeting this pathway for cancer treatment. Finally, the efficacy of reserpine as an adjuvant/neoadjuvant therapy for metastasis in vivo provides proof-of-principle for PMN targeting. However, because patients can experience hypotension during chemotherapy (Senkus and Jassem, 2011Senkus E. Jassem J. Cardiovascular effects of systemic cancer treatment.Cancer Treat. Rev. 2011; 37: 300-311Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), caution should be exercised when considering adjuvant treatment with reserpine. Nevertheless, reserpine and other molecules targeting TEV uptake should be further characterized for their ability to inhibit PMNs and metastasis in additional preclinical mouse models of cancer, such as breast and pancreatic carcinomas, to better understand the broad utility of targeting this pathway for treating metastasis. The authors wish to thank Virginia Pascual and Robert Schwartz for helpful reading of the manuscript and providing insightful feedback. An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular VesiclesOrtiz et al.Cancer CellJanuary 14, 2019In BriefOrtiz et al. reveal that melanoma-derived extracellular vesicles (EV) downregulate type I interferon receptor (IFNAR1) and cholesterol 25-hydroxylase (CH25H) in normal cells to facilitate EV uptake and pre-metastatic niche development. IFNAR1-CH25H upregulation or reserpine treatment inhibits EV uptake and metastasis. Full-Text PDF Open Archive