Arresting the Colonial Destiny of Metastatic Seeds with DNA Aptamers
Authors: Gerolama Condorelli, Paloma H Giangrande, Vittorio de Franciscis
Journal: Molecular Therapy (2015)

In recent years, several studies have identified a class of carbohydrate-binding proteins known as (endothelial) E- and (platelet) P-selectins as potential therapeutic targets for inhibiting hematogenous tumor metastasis, owing to their critical role in the metastatic process. Selectins enable tethering and rolling of circulating tumor cells or cancer stem cells (CTC/CSCs) to the postcapillary venules, promoting their transmigration through the endothelium and subsequent homing to metastatic sites. In this issue of Molecular Therapy, Kang et al. describe the preclinical characterization of a nucleic acid DNA aptamer to E-selectin as a safe and effective therapeutic option for preventing this process and inhibiting breast cancer metastasis.1Kang S-A Hasan N Mann AP Zheng W Zhao L Morris L et al.Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.Mol Ther. 2015; 23: 1044-1054Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar One of the causes of cancer recurrence and therapy failure is the ability of cancer cells to leave their local surroundings and disseminate to distal sites by migrating through either lymphatic vessels or the bloodstream (Figure 1). Cancer metastasis is a multistep process where cancer cells degrade the extracellular matrix and break away from the primary tumors.2Chiang AC Massague J Molecular basis of metastasis.N Engl J Med. 2008; 359: 2814-2823Crossref PubMed Scopus (799) Google Scholar This process is initiated by the “metamorphosis” of a subset of primary tumor cells of epithelial origin via an epithelial-to-mesenchymal transformation that bestows on them key characteristics of mesenchymal cells, such as enhanced motility and plasticity. This enables them to enter the bloodstream (a process known as intravasation), at which point they are typically referred to as CTC/CSCs. To survive, hematogenous CTC/CSCs must escape the bloodstream (extravasation) and reach distant niches favorable to their growth. The first critical step of extravasation is the rolling of CTC/CSCs on the luminal surface of endothelial cells. This process is mediated, on the endothelial cell side, by a class of carbohydrate-binding proteins known as E- and P-selectins. In breast cancer, this phenomenon may be hormone-dependent, possibly depending on estrogen or progesterone receptor status. Aptamers are single-chain DNA (or RNA) oligonucleotides whose binding properties depend on their sequence and three-dimensional structure. They are identified from high-complexity DNA or RNA sequence libraries through an iterative process known as systematic evolution of ligands by exponential enrichment (SELEX).3Blind M Blank M Aptamer selection technology and recent advances.Mol Ther Nucleic Acids. 2014; 4: e223Abstract Full Text Full Text PDF Scopus (216) Google Scholar Aptamers are typically selected for properties such as high affinity and specific binding to a target protein, and have binding affinities and specificities comparable to those of antibody–antigen interactions. Aptamers have been developed for a variety of diagnostic and therapeutic applications.4Thiel KW Giangrande PH Therapeutic applications of DNA and RNA aptamers.Oligonucleotides. 2009; 19: 209-222Crossref PubMed Scopus (136) Google Scholar,5Keefe AD Pai S Ellington A Aptamers as therapeutics.Nat Rev Drug Discov. 2010; 9: 537-550Crossref PubMed Scopus (1258) Google Scholar The latter involve their tendency to inhibit their protein targets upon binding. Therapeutic aptamers are typically modified with fluoro or O-methyl groups at the 2′ position of their sugar moiety, which renders them resistant to degradation by serum nucleases and reduces their immunogenicity.4Thiel KW Giangrande PH Therapeutic applications of DNA and RNA aptamers.Oligonucleotides. 2009; 19: 209-222Crossref PubMed Scopus (136) Google Scholar,5Keefe AD Pai S Ellington A Aptamers as therapeutics.Nat Rev Drug Discov. 2010; 9: 537-550Crossref PubMed Scopus (1258) Google Scholar Aptamers can also be modified after selection (e.g., conjugation of high-molecular-weight polyethylene glycol or cholesterol) to improve their pharmacokinetics and pharmacodynamics.4Thiel KW Giangrande PH Therapeutic applications of DNA and RNA aptamers.Oligonucleotides. 2009; 19: 209-222Crossref PubMed Scopus (136) Google Scholar,5Keefe AD Pai S Ellington A Aptamers as therapeutics.Nat Rev Drug Discov. 2010; 9: 537-550Crossref PubMed Scopus (1258) Google Scholar Therefore, aptamers are emerging as promising therapeutic reagents that combine a high specificity of target recognition, typical of monoclonal antibodies, with the additional advantage of a streamlined production process that does not make use of animal cells, and is thus relatively rapid with high batch-to-batch fidelity.6Cerchia L de Franciscis V Targeting cancer cells with nucleic acid aptamers.Trends Biotechnol. 2010; 28: 517-525Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar,7Zhou J Rossi JJ Cell-type-specific, aptamer-functionalized agents for targeted disease therapy.Mol Ther Nucleic Acids. 2014; 3: e169Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar To date, monoclonal antibodies,8Gakhar G Navarro VN Jurish M Lee GY Tagawa ST Akhtar NH et al.Circulating tumor cells from prostate cancer patients interact with E-selectin under physiologic blood flow.PLoS One. 2013; 8: e85143Crossref PubMed Scopus (33) Google Scholar modified aptamers,9Faryammanesh R Lange T Magbanua E Haas S Meyer C Wicklein D et al.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. 2014; 9: e93173Crossref PubMed Scopus (26) Google Scholar,10Mai J Huang Y Mu C Zhang G Xu R Guo X et al.Bone marrow endothelium-targeted therapeutics for metastatic breast cancer.J Control Release. 2014; 187: 22-29Crossref PubMed Scopus (44) Google Scholar,11Mann AP Somasunderam A Nieves-Alicea R Li X Hu A Sood AK et al.Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.PLoS One. 2010; 5 (pii. E): 13050Crossref PubMed Scopus (72) Google Scholar and glycomimetic antagonists (GMI-1359; GlycoMimetics) have been evaluated as E- or P-selectin inhibitors. Kang et al. report, for the first time, the use of a DNA aptamer as a safe and effective alternative to block binding of CD44 to E-selectin and inhibit adhesion of estrogen receptor–negative (ER–) breast cancer cells to endothelial cells, extravasation, and premetastatic niche formation. First, the authors used flow adhesion and transendothelial migration assays to investigate the involvement of E-selectin in hematogenous metastasis. The authors show that E-selectin expression results in a twofold increase in shear-resistant adhesion of several aggressive breast cancer cell lines (e.g., ER–, MDA-MB-231, and MDA-MB-468), but not ER+ breast cancer cell lines (considered less aggressive) to endothelial cells (HMVECs). In addition, E-selectin expression in HMVECs was sufficient to induce the transendothelial migration of ER–, but not of ER+ cells. These results demonstrate that E-selectin is sufficient to promote the adhesion of ER– breast cancer cells to endothelial cells. Based on these findings, the authors reasoned that E-selectin protein binding partners must be preferentially expressed on ER– cells. A candidate binding partner for E-selectin is the cell-surface glycoprotein CD44 antigen, implicated in cell adhesion and migration. Not surprisingly, CD44 is reported to be highly expressed on the surface of breast cancer stem cells.12Gong J Weng D Eguchi T Murshid A Sherman MY Song B Calderwood SK Targeting the hsp70 gene delays mammary tumor initiation and inhibits tumor cell metastasis.Oncogene. 2015; (e-pub ahead of print 9 February 2015)Google Scholar Importantly, Kang et al. demonstrate that CD44 binds directly to E-selectin and that its expression is required for E-selectin-induced shear-resistant adhesion of ER– breast cancer cells. Furthermore, expression of CD44 is higher in distant metastases than in localized tumors, and higher in biopsy specimens from ER– patients than in those from ER+ metastatic breast cancer patients. By contrast, the authors observed elevated E-selectin expression in carcinoma tissues and noncancerous lung tissues adjacent to metastatic lung lesion, which suggests that blocking binding of 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.10Mai J Huang Y Mu C Zhang G Xu R Guo X et al.Bone marrow endothelium-targeted therapeutics for metastatic breast cancer.J Control Release. 2014; 187: 22-29Crossref PubMed Scopus (44) Google Scholar,11Mann AP Somasunderam A Nieves-Alicea R Li X Hu A Sood AK et al.Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.PLoS One. 2010; 5 (pii. E): 13050Crossref PubMed Scopus (72) Google Scholar 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 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.13Ghajar CM Metastasis prevention by targeting the dormant niche.Nat Rev Cancer. 2015; 15: 238-247Crossref PubMed Scopus (218) Google Scholar 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.14Hong MK Macintyre G Wedge DC Van Loo P Patel K Lunke S et al.Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer.Nat Commun. 2015; 6: 6605Crossref PubMed Scopus (260) Google Scholar The use of aptamers offers an invaluable tool for better elucidating these processes and for developing much-needed therapies against metastatic disease.