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commentary CD44 to E-selectin may represent a viable therapeutic option for preventing distal metastases. To block the shear-resistant adhesion of CTCs within the premetastatic niche, the authors developed an antagonistic thiophosphate backbone–modified DNA aptamer against E-selectin (ESTA) that displayed greater affinity for E-selectin compared to the E-selectin natural ligand.10,11 They went on to show that the ESTA thioaptamer, but not a control aptamer, inhibited binding of CD44 to E-selectin, resulting in potent inhibition of the shear-resistant adhesion of ER–/CD44+ (MDA-MB-231) cells to endothelial HMVECs. On the basis of the E-selectin–specific antagonistic effect of ESTA in vitro, the authors evaluated the antimetastatic effect of the ESTA aptamer in vivo and found that a single bolus injection of the aptamer reduced formation of ER– 4T1-derived hematogenous metastases by 92.2%. 4T1 is a highly metastatic breast murine cancer cell line that can be transplanted into immunocompetent syngeneic Balb/C mice. ESTA’s effects were evident only in preconditioned mice subjected to intraperitoneal injections of conditioned medium containing vascular endothelial growth factor, which is necessary for E-selectin expression. Homing of CD44-expressing breast cancer cells in the circulation was also inhibited with a single injection of ESTA through the inhibition of E-selectin. By contrast, inhibition of metastasis was not observed when a similar amount of control random aptamer was injected into wild-type Balb/C mice or when ESTA was injected into E-selectin knockout mice. These observations suggest that ESTA exerts its effect through functional blockade of E-selectin. Importantly, the reduction of lung metastasis did not result in a relocation of metastasis to other sites (e.g., brain) indicating that ESTA inhibits cancer cell homing and hematogenous metastasis by body-wide E-selectin inhibition. Because the ESTA aptamer can bind to both human and mouse E-selectins, the same aptamer can be evaluated in preclinical animal models of disease and in future clinical trials in humans, thereby expediting its clinical translation. Finally, ESTA appeared to have no

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cytotoxic effect and thus should be safe for use in humans. The short half-life of ESTA requires daily injections to achieve desirable efficacy in vivo—a potential limitation for clinical application. More work is needed to improve the circulating half-life (T1/2) and improve potency of ESTA. Moreover, the ESTA aptamer was only effective at inhibiting dissemination of ER–/CD44-expressing cells and not ER+ cells, thus precluding ER+ patients from benefiting from this therapy. Although additional preclinical studies are needed before this therapy can be used in patients with ER– breast cancers, this work highlights the use of aptamers for targeting homing of cancer cells to distant sites and thus represents a blueprint for developing aptamer-based therapeutics for the prevention of metastasis.13 By targeting the vessel surface of the premetastatic niche, the approach described by Kang et al. opens the way to an innovative and personalized strategy for precise therapeutic intervention in patients with advanced cancers. Furthermore, seeding to the premetastatic niche is a complex and still poorly understood mechanism that probably involves the synergistic cooperation of many distinct tumor subclones for the promotion of mutual growth and survival.14 The use of aptamers offers an invaluable tool for better elucidating these processes and for developing much-needed therapies against metastatic disease.

REFERENCES 1. Kang, S-A, Hasan, N, Mann, AP, Zheng, W, Zhao, L, Morris, L et al. (2015). Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis. Mol Ther 23: 1044–1054. 2. Chiang, AC and Massague, J (2008). Molecular basis of metastasis. N Engl J Med 359: 2814–2823. 3. Blind, M and Blank, M (2014). Aptamer selection technology and recent advances. Mol Ther Nucleic Acids 4: e223. 4. Thiel, KW and Giangrande, PH (2009). Therapeutic applications of DNA and RNA aptamers. Oligonucleotides 19: 209–222. 5. Keefe, AD, Pai, S and Ellington, A (2010). Aptamers as therapeutics. Nat Rev Drug Discov 9: 537–550. 6. Cerchia, L and de Franciscis, V (2010). Targeting cancer cells with nucleic acid aptamers. Trends Biotechnol 28: 517–525. 7. Zhou, J and Rossi, JJ (2014). Cell-type-specific, aptamer-functionalized agents for targeted disease therapy. Mol Ther Nucleic Acids 3: e169. 8. Gakhar, G, Navarro, VN, Jurish, M, Lee, GY, Tagawa, ST, Akhtar, NH et al. (2013). Circulating tumor cells from prostate cancer patients interact with E-selectin under physiologic blood flow. PLoS One 8: e85143. 9. Faryammanesh, R, Lange, T, Magbanua, E, Haas, S, Meyer, C, Wicklein, D et al. (2014). SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation. PLoS One 9: e93173. 10. Mai, J, Huang, Y, Mu, C, Zhang, G, Xu, R, Guo, X et al. (2014). Bone marrow endothelium-targeted therapeutics for metastatic breast cancer. J Control Release 187: 22–29. 11. Mann, AP, Somasunderam, A, Nieves-Alicea, R, Li, X, Hu, A, Sood, AK et al. (2010). Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting. PLoS One 5: pii. E13050. 12. Gong, J, Weng, D, Eguchi, T, Murshid, A, Sherman, MY, Song, B and Calderwood, SK (2015). Targeting the hsp70 gene delays mammary tumor initiation and inhibits tumor cell metastasis. Oncogene; e-pub ahead of print 9 February 2015. 13. Ghajar, CM (2015). Metastasis prevention by targeting the dormant niche. Nat Rev Cancer 15: 238–247. 14. Hong, MK, Macintyre, G, Wedge, DC, Van Loo, P, Patel, K, Lunke, S et al. (2015). Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer. Nat Commun 6: 6605.

RNA Mimics as Therapeutics for Cardiac Regeneration: A Paradigm Shift Mauro Giacca1 doi:10.1038/mt.2015.86 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy Correspondence:  Mauro Giacca, Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Padriciano 99, 34149 Trieste, Italy. E-mail: [email protected]

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hrough an elegant combination of transgenic techniques, a study by Tian et al. recently published in Science Translational Medicine1 shows that a cluster of five microRNAs (miRNAs), miR-302/367, controls cardiac myocyte proliferation during embryonic and

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neonatal life. More notably, reactivation of these miRNAs following myocardial infarction promotes re-entry of adult cardiomyocytes into the cell cycle and induces cardiac regeneration. The authors found that these miRNAs interfere with the recently described Hippo pathway that regulates proliferation of various cell types, including cardiomyocytes. More specifically, miR-302/367 directly reduces the expression of three inhibitory proteins in this pathway, leading to the activation of the Yap1 transcription factor, a master regulator of cardiac proliferation. The notion that miRNAs control cardiomyocyte proliferation is not entirely novel. Over the past few years, there has been a flood of exciting evidence that the miRNA network plays an essential role in regulating the extent of cardiomyocyte replication during development and that it might be harnessed in adult hearts to promote repair after myocardial damage. The sudden—and still, for the most part, not understood— arrest of cardiomyocyte proliferation immediately after birth correlates with increased levels of a large set of miRNAs, some of which are causally involved in this event. These include the six members of the miR-15 family2 and miR-29a,3 which target essential components of the cell cycle and checkpoint machineries; inhibition of miR-15 expands the proliferative potential of cardiomyocytes during fetal and early postnatal life and stimulates regeneration after myocardial infarction.4,5 By contrast, several miRNAs act in an opposite manner, by stimulating cardiomyocyte proliferation. A genomic-scale, highthroughput screen performed in my laboratory has shown that at least 40 miRNAs encoded by the human genome are capable of stimulating proliferation of neonatal cardiomyocytes.6 Among these miRNAs are m ­ iR-199a-3p, ­miR-590–3p, and two large families of miRNAs that previous work had shown to be essential in the regulation of embryonic cell proliferation: the miR-17/92 and the miR-302/367 clusters.7 Members of both families are highly expressed in em-

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commentary bryonic stem cells and are essential to maintain the undifferentiated state.8 Transgenic overexpression of both the miR-17/92 cluster9 and the miR302/367 cluster, as shown by Tian et al.,1 induces significant expansion of the cardiomyocyte pool during embryonic life, whereas their knockout leads to hypoplasia and cardiac dysfunction. More notably, in both cases reactivation of cardiomyocyte proliferation in adult mice is capable of increasing the usually ineffectual attempt at cardiac regeneration that is observed after myocardial infarction. The results of the new study strengthen and significantly expand a paradigm shift that has been occurring in the cardiovascular field over the last few years, namely that cardiac regeneration after damage can be achieved by stimulating the proliferation of already existing cardiomyocytes rather than having to rely on the implantation of exogenous cells. The notion that cardiomyocytes, or at least a subset of these, are endowed with an intrinsic capacity to proliferate during the adult life is consistent with earlier observations that a limited regenerative attempt normally occurs after infarction,10 that almost 50% of the myocardium is recycled during a lifetime,11 and that cardiac cell turnover physiologically occurs at a magnitude of ~1% cells per year.12 Expanding the endogenous cardiomyocyte proliferation capacity by genetic drugs thus appears to represent an exciting and more sustainable approach to achieve cardiac regeneration compared to ex vivo expansion and implantation of stem cell–derived cardiomyocytes. In this respect, it is worth recalling that cardiac regeneration in zebrafish, which occurs through the partial dedifferentiation and proliferation of preexisting cardiomyocytes,13,14 is also regulated by the miRNA network. In particular, recent evidence shows that miR-99/100 and Let-7a/c are downregulated during the regeneration process in zebrafish and that their forced inhibition is also effective in promoting regeneration in the mouse heart.15

Another merit of the article by Tian et al. is the description of a molecular mechanism for the miR-302/367 proliferative action based on its effect on the Hippo pathway. Originally discovered by genetic screens in the Drosophila eye, this pathway is a broad and essential regulator of cell proliferation and organ size. In mammalian cells, the main effector of the pathway is the transcriptional coactivator Yap. The kinase Mst1/2 (Hippo in Drosophila) interacts with WW45 to phosphorylate and activate the Lats1/2 and Mob1 complex, which in turn phosphorylates and inactivates Yap. In a crescendo of findings over the past few years, different investigators have shown that embryonic knockout of Mst1, WW45, and Lats causes cardiac hyperplasia, whereas overexpression of Mst1 and Lats, or knockout of Yap, leads to hypoplasia and cardiac dilation (reviewed in ref. 16). The pathway also appears to be active during postnatal life: in transgenic mice that overexpress a constitutively active Yap mutant, myocardial infarction is repaired with reduced fibrosis and increased myocardial tissue formation;17 consistent with this, Hippo deficiency enhances cardiomyocyte generation.18 Thus, the Hippo pathway stands as a master regulator of cardiac cell proliferation during both embryogenesis and postnatal life. The new study strengthens this conclusion by showing that at least one of the mechanisms of action of the miR302/367 cluster is through repression of the kinases Mst1 and Lats2/Mob1b. Whether other miRNAs with the capacity to stimulate cardiomyocyte proliferation also act through the Hippo pathway remains a very interesting question that deserves further investigation. A most striking finding by Tian et al. is the demonstration that cardiac regeneration after myocardial infarction could be achieved by the administration, once a day, of synthetic miRNA mimics in a neutral lipid formulation. This finding is truly groundbreaking because, in the field of small-RNA therapeutics, it was thought that miRNA mimics would find little therapeutic usefulness because of

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commentary their short half-life. This misconception arises because miRNA mimics, in contrast to miRNA inhibitors, must maintain their native chemical structure so as to be properly recognized by the RNA interference machinery, and thus could not be chemically modified to increase stability. The finding that the miR302b/c mimics are effective following systemic administration paves the way to a number of other mimic applications within and beyond the cardiovascular field, without the need to rely on viral vectors for intracellular miRNA expression. Will the miR302b/c mimics themselves become a real drug for patients with myocardial infarction? This is difficult to predict at this moment, but the biological activity of this miRNA cluster in inducing massive dedifferentiation toward an embryonic stem cell phenotype,19 and thus its deleterious effects when expressed for prolonged periods, would suggest caution. Should other miRNAs be identified that are equally effective in acting through the Hippo pathway but less prone to induce massive cell dedifferentiation, these would probably be better suited to development into human therapeutics. Despite this ­caveat, Tian et al. provide formidable proof-of-concept evidence that organ ­regeneration in vivo can be attained by

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exogenously delivered small-RNA drugs— certainly a welcome possibility, given the current critical burden of degenerative diseases. ACKNOWLEDGMENTS

This work was supported by Advanced Grant 250124 from the European Research Council. The author is grateful to Suzanne Kerbavcic for valuable editorial assistance.

REFERENCES

1. Tian, Y, Liu, Y, Wang, T, Zhou, N, Kong, J, Chen, L et al. (2015). A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice. Sci Transl Med 7: 279ra38. 2. Porrello, ER, Johnson, BA, Aurora, AB, Simpson, E, Nam, YJ, Matkovich, SJ et al. (2011). MiR-15 family regulates postnatal mitotic arrest of cardiomyocytes. Circ Res 109: 670–679. 3. Cao, X, Wang, J, Wang, Z, Du, J, Yuan, X, Huang, X et al. (2013). MicroRNA profiling during rat ventricular maturation: a role for miR-29a in regulating cardiomyocyte cell cycle re-entry. FEBS Lett 587: 1548–1555. 4. Porrello, ER, Mahmoud, AI, Simpson, E, Johnson, BA, Grinsfelder, D, Canseco, D et al. (2013). Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proc Natl Acad Sci USA 110: 187–192. 5. Hullinger, TG, Montgomery, RL, Seto, AG, Dickinson, BA, Semus, HM, Lynch, JM et al. (2012). Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res 110: 71–81. 6. Eulalio, A, Mano, M, Dal Ferro, M, Zentilin, L, Sinagra, G, Zacchigna, S et al. (2012). Functional screening identifies miRNAs inducing cardiac regeneration. Nature 492: 376–381. 7. Tiscornia, G and Izpisúa Belmonte, JC (2010). MicroRNAs in embryonic stem cell function and fate. Genes Dev 24: 2732–2741. 8. Barroso-del Jesus, A, Lucena-Aguilar, G and Menendez, P (2009). The miR-302–367 cluster as a potential stemness regulator in ESCs. Cell Cycle 8:

394–398. 9. Chen, J, Huang, ZP, Seok, Hy, Ding, J, Kataoka, M, Zhang, Z et al. (2013). miR-17–92 cluster is required for and sufficient to induce cardiomyocyte proliferation in postnatal and adult hearts. Circ Res 112: 1557–1566. 10. Beltrami, AP, Urbanek, K, Kajstura, J, Yan, SM, Finato, N, Bussani, R et al. (2001). Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 344: 1750–1757. 11. Bergmann, O, Bhardwaj, RD, Bernard, S, Zdunek, S, Barnabé-Heider, F, Walsh, S et al. (2009). Evidence for cardiomyocyte renewal in humans. Science 324: 98–102. 12. Senyo, SE, Steinhauser, ML, Pizzimenti, CL, Yang, VK, Cai, L, Wang, M et al. (2013). Mammalian heart renewal by pre-existing cardiomyocytes. Nature 493: 433–436. 13. Kikuchi, K, Holdway, JE, Werdich, AA, Anderson, RM, Fang, Y, Egnaczyk, GF et al. (2010). Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature 464: 601–605. 14. Jopling, C, Sleep, E, Raya, M, Marti, M, Raya, A and Izpisúa Belmonte, JC (2010). Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464: 606–609. 15. Aguirre, A, Montserrat, N, Zacchigna, S, Nivet, E, Hishida, T, Krause, MN et al. (2014). In vivo activation of a conserved microRNA program induces mammalian heart regeneration. Cell Stem Cell 15: 589–604. 16. Lin, Z and Pu, WT (2014). Harnessing Hippo in the heart: Hippo/Yap signaling and applications to heart regeneration and rejuvenation. Stem Cell Res 13: 571–581. 17. Xin, M, Kim, Y, Sutherland, LB, Murakami, M, Qi, X, McAnally, J et al. (2013). Hippo pathway effector Yap promotes cardiac regeneration. Proc Natl Acad Sci USA 110: 13839–13844. 18. Heallen, T, Morikawa, Y, Leach, J, Tao, G, Willerson, JT, Johnson, RL et al. (2013). Hippo signaling impedes adult heart regeneration. Development 140: 4683–4690. 19. Anokye-Danso, F, Trivedi, CM, Juhr, D, Gupta, M, Cui, Z, Tian, Y et al. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8: 376–388.

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