On the Mechanism and Benefit of siRNA-mediated Targeting of CD47 in Cancer
Authors: Xiwen Zhao, Hanke Matlung, Taco W Kuijpers, Timo K van den Berg
Journal: Molecular Therapy (2013)

To the editor: We have read with interest the report by Wang et al.1Wang Y Xu Z Guo S Zhang L Sharma A Robertson GP et al.Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.Mol Ther. 2013; 21: 1919-1929Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar in this issue, “Intravenous Delivery of siRNA Targeting CD47 Effectively Inhibits Melanoma Tumor Growth and Lung Metastasis,” describing the targeting of CD47 in melanoma with short interfering RNA (siRNA). In a well-designed series of experiments, the investigators show that both in vitro and in vivo delivery to melanoma cells of CD47 siRNA encapsulated in nanoparticles results in a potent and selective reduction in cancer cell CD47 expression and causes a strong macrophage-mediated elimination of the tumor cells. In particular, they show that intravenous injection of the CD47 siRNA-containing nanoparticles reduces the growth and metastasis of either locally (subcutaneously) or systemically (intravenously) injected B16F10 melanoma cells in syngeneic mice by >90% and >70%, respectively. They propose that CD47, which is widely expressed on essentially all normal cells and cancer cells, acts as a “don’t-eat-me” signal that interacts with the inhibitory immunoreceptor SIRPα expressed on macrophages and other myeloid cells to prevent phagocytosis. However, we think that, given other available evidence, there might be alternative explanations for these results. Our own studies—using the same metastatic B16F10 melanoma model performed in mice that lack the cytoplasmic tail of SIRPα responsible for inhibitory signaling—have demonstrated that SIRPα signaling is completely dispensable for melanoma metastasis and outgrowth.2Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (221) Google Scholar Interestingly, when the mice were also treated with therapeutic antibodies directed against the melanoma cells, SIRPα signaling was found to restrict the antibody-dependent destruction of the cancer cells.2Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (221) Google Scholar Moreover, in vitro experiments employing SIRPα-expressing human neutrophils or monocytes as effector cells in antibody-dependent cellular cytotoxicity experiments demonstrated that interference with CD47–SIRPα interactions, either by short hairpin RNA–mediated knockdown of CD47 in the tumor cells or by using antagonistic antibodies against CD47 or SIRPα, potentiated the antibody-mediated killing of the cancer cells but did not affect cytotoxicity in the absence of the tumor-targeting therapeutic antibody.2Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (221) Google Scholar Finally, a recent study by Weiskopf et al.3Weiskopf K Ring AM Ho CC Volkmer JP Levin AM Volkmer AK et al.Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies.Science. 2013; 341: 88-91Crossref PubMed Scopus (325) Google Scholar confirmed that antagonizing the CD47–SIRPα interaction has, at least in most cancer types, only minimal in vivo antitumor effects in the absence of cancer therapeutic antibodies but is strongly synergistic in combination with several US Food and Drug Administration–approved cancer therapeutic antibodies directed against different tumor cells. Therefore, we believe that Wang and colleagues’ results cannot be explained simply by a lack of inhibitory signaling via the CD47–SIRPα axis. One potential explanation for the observed discrepancy is that the nanoparticles that Wang et al. used may not only have acted as a vehicle for siRNA delivery but also may have opsonized the tumor cells for recognition by phagocytes, which could have directly triggered phagocytosis and killing by macrophages and/or neutrophils, or may even have promoted T cell–mediated immunity against the tumors as recently reported.4Tseng D Volkmer JP Willingham SB Contreras-Trujillo H Fathman JW Fernhoff NB et al.Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.Proc Natl Acad Sci USA. 2013; 110: 11103-11108Crossref PubMed Scopus (396) Google Scholar Several studies published in recent years have suggested that targeting of human CD47 in xenogeneic tumor models using monoclonal antibodies known to block CD47–SIRPα interactions is sufficient to provide strong antitumor effects.5Majeti R Chao MP Alizadeh AA Pang WW Jaiswal S Gibbs Jr KD et al.CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.Cell. 2009; 138: 286-299Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar,6Jaiswal S Jamieson CH Pang WW Park CY Chao MP Majeti R et al.CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.Cell. 2009; 138: 271-285Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar,7Chao MP Alizadeh AA Tang C Myklebust JH Varghese B Gill S et al.Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma.Cell. 2010; 142: 699-713Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar,8Chao MP Alizadeh AA Tang C Jan M Weissman-Tsukamoto R Zhao F et al.Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia.Cancer Res. 2011; 71: 1374-1384Crossref PubMed Scopus (286) Google Scholar,9Chao MP Tang C Pachynski RK Chin R Majeti R Weissman IL Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy.Blood. 2011; 118: 4890-4901Crossref PubMed Scopus (145) Google Scholar,10Willingham SB Volkmer JP Gentles AJ Sahoo D Dalerba P Mitra SS et al.The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.Proc Natl Acad Sci USA. 2012; 109: 6662-6667Crossref PubMed Scopus (997) Google Scholar However, as we and others have previously argued,11Zhao XW Kuijpers TW van den Berg TK Is targeting of CD47-SIRPα enough for treating hematopoietic malignancy.Blood. 2012; 119 (author reply 4334–4335): 4333-4334Crossref PubMed Scopus (28) Google Scholar,12Zhao XW Matlung HL Kuijpers TW van den Berg TK On the mechanism of CD47 targeting in cancer.Proc Natl Acad Sci USA. 2012; 109 (E2843 author reply): E2844-2845Crossref Scopus (7) Google Scholar,13Soto-Pantoja DR Miller TW Frazier WA Roberts DD Inhibitory signaling through signal regulatory protein-α is not sufficient to explain the antitumor activities of CD47 antibodies.Proc Natl Acad Sci USA. 2012; 109 (E2842 author reply): E2844-E2845Crossref Scopus (24) Google Scholar these in vivo studies employed a CD47-specific monoclonal antibody (i.e., clone B6H12) that can itself trigger phagocyte effector functions,2Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (221) Google Scholar,11Zhao XW Kuijpers TW van den Berg TK Is targeting of CD47-SIRPα enough for treating hematopoietic malignancy.Blood. 2012; 119 (author reply 4334–4335): 4333-4334Crossref PubMed Scopus (28) Google Scholar and this might explain, at least in part, the effects of CD47-specific antibodies on tumor growth in vivo. Regardless of the underlying mechanism(s), the approach of Wang et al. could still, of course, be a very promising strategy for treatment of human cancer patients. The apparent high level of selectivity (i.e., the tropism) of nanoparticle-mediated delivery of CD47 siRNA to tumor cells and the limited cytotoxicity to the recipient are both attractive features. However, the authors also provide evidence that the siRNA-mediated CD47 knockdown enhances anchorage-independent growth of melanoma cells in vitro and substantially increases in vivo growth of tumors in the absence of macrophages.1Wang Y Xu Z Guo S Zhang L Sharma A Robertson GP et al.Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.Mol Ther. 2013; 21: 1919-1929Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar The latter evidence is reminiscent of the cell-autonomous protective role of CD47 against ionizing radiation identified in reports studying CD47 deficiency or knockdown in mice.14Maxhimer JB Soto-Pantoja DR Ridnour LA Shih HB DeGraff WG Tsokos M et al.Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling.Sci Transl Med. 2009; 1: 3ra7Crossref PubMed Scopus (114) Google Scholar,15Soto-Pantoja DR Miller TW Pendrak ML DeGraff WG Sullivan C Ridnour LA et al.CD47 deficiency confers cell and tissue radioprotection by activation of autophagy.Autophagy. 2012; 8: 1628-1642Crossref PubMed Scopus (67) Google Scholar,16Soto-Pantoja DR Ridnour LA Wink DA Roberts DD Blockade of CD47 increases survival of mice exposed to lethal total body irradiation.Sci Rep. 2013; 3: 1038Crossref PubMed Scopus (48) Google Scholar Therefore, it seems that CD47 plays multiple opposing roles in cancer cells, and the question with respect to CD47 siRNA targeting in patients is whether the beneficial effects will be able to outweigh the tumor-promoting ones.