Targeting Axl with an high-affinity inhibitory aptamer

Abstract

Axl is a tyrosine kinase receptor that was first identified as a transforming gene in human myeloid leukemia. Recent converging evidence suggests its implication in cancer progression and invasion for several solid tumors, including lung, breast, brain, thyroid, and pancreas. In the last decade, Axl has thus become an attractive target for therapeutic development of more aggressive cancers. An emerging class of therapeutic inhibitors is now represented by short nucleic acid aptamers. These molecules act as high affinity ligands with several advantages over conventional antibodies for their use in vivo, including their small size and negligible immunogenicity. Furthermore, these molecules can easily form conjugates able to drive the specific delivery of interfering RNAs, nanoparticles, or chemotherapeutics. We have thus generated and characterized a selective RNA-based aptamer, GL21.T that binds the extracellular domain of Axl at high affinity (12 nmol/l) and inhibits its catalytic activity. GL21.T blocked Axl-dependent transducing events in vitro, including Erk and Akt phosphorylation, cell migration and invasion, as well as in vivo lung tumor formation in mice xenografts. In this respect, the GL21.T aptamer represents a promising therapeutic molecule for Axl-dependent cancers whose importance is highlighted by the paucity of available Axl-specific inhibitory molecules.

Figures

Figure 1

GL21.T aptamer specifically interacts with Axl. (a) Binding isotherm for GL21.T: EC-Axl (left) and GL21.T:EC-Dtk (right) complexes. (b) EC-Axl or EC-Dtk (40 nmol/l, with and without 5 mmol/l DTT treatment), were incubated with 1 nmol/l GL21.T, protein-bound RNA was collected by nitrocellulose filters and radioactivity quantified. (c) Left, binding of 50 nmol/l radiolabeled GL21.T on the indicated cell lines. Right, lysates from the indicated cell lines were immunoblotted with anti-Axl antibodies. (d), Left, binding of 50 nmol/l radiolabeled GL21 on U87MG, SkBr3, or SkBr3 cells following 72 hours-transfection with Axl TruClone (Axl). Right, lysates from SKBr3 or SKBr3 transfected with Axl were immunoblotted with anti-Axl antibodies. (e), Left, binding of 50 nmol/l radiolabeled GL21.T on U87MG cells following 72 hours-transfection with a specific Axl short hairpin RNA (shRNA) (shRNAAxl) or a nonrelated shRNA (shRNActrl). Right, lysates from U87MG cells following 72 hours-transfection with shRNAAxl or shRNActrl were immunoblotted with anti-Axl antibodies. Values below the blot indicate signal levels relative to shRNActrl-transfected cells, arbitrarily set to 1 (labeled with asterisk). Intensity of bands has been calculated using the NIH Image Program on at least two different expositions to assure the linearity of each acquisition. In (c–e), blots shown are representative of at least three independent experiments and anti-α–tubulin antibodies were used as an internal control. (f) Binding of 50 nmol/l radiolabeled GL21.T, prior incubated with 150 nmol/l EC-Axl for 15 minutes at 37 °C, on U87MG cells. In (b–f), the results are expressed relative to the background binding detected with the unrelated aptamer used as a negative control. (g) Internalization rate of GL21.T and unrelated uptamer. Results are expressed as percentage of internalized RNA relative to total bound aptamer. In (a–g), error bars depict means ± SD (n = 3).

Figure 2

GL21.T inhibits Axl activation. (a) Serum-starved U87MG and A549 cells were either left untreated or treated for 3 hours with 200 nmol/l GL21.T or the unrelated aptamer and then stimulated for the indicated times with Gas6 in the presence of each aptamer. Cell lysates were either immunoprecipitated with anti-(phospho)-tyrosine (pTyr) antibodies and immunoblotted with anti-Axl antibodies or immunoblotted with anti-Axl, anti-(phospho)-Erk1/2 (pErk), anti-(phospho)-Akt (pAkt) antibodies, as indicated. Filters were stripped and reprobed with anti-Erk and anti-Akt antibodies, as indicated. Values below the blots indicate signal levels relative to 15 minutes-Gas6 stimulated unrelated aptamer control, arbitrarily set to 1 (labeled with asterisk). Quantitations were done as in Figure 1. Blots shown are representative of at least four independent experiments. (b) Indicated cell lines were left untreated or treated for 24 hours with increasing concentrations of GL21.T or the unrelated aptamer (as indicated), cell viability was analyzed as reported in Materials and Methods and expressed as percent of viable treated cells with respect to control, untreated cells. (c) Left, A549 cells were treated for 72 hours with GL21.T or the unrelated aptamer (200 nmol/l-final concentration) or A549 were transfected with shRNAAxl or shRNActrl and cell viability was analyzed as in b. Right, lysates from A549 cells following 72 hours-transfection with shRNAAxl or shRNActrl were immunoblotted with anti-Axl antibodies. Values below the blot indicate signal levels relative to mock-transfected cells, arbitrarily set to 1 (labeled with asterisk). Quantitation was done as in Figure 1. (d) U87MG and A549 cells were treated for 24 or 48 hours with GL21.T or the unrelated aptamer (200 nmol/l -final concentration) and proliferation was determined by [3H]-thymidine incorporation. (e) A549 cells were treated for 24 hours with GL21.T or the unrelated aptamer (200 nmol/l-final concentration) as single agents or in combination with TRAIL, cisplatin (Cis) and paclytaxel (Pacl) at the indicated concentrations. Cell viability was analyzed as in b. (f) A549 cells were treated for 24 hours with GL21.T or the unrelated aptamer (200 nmol/l-final concentration) as single agents or in combination with CL4 aptamer at the indicated concentrations. Cell viability was analyzed as in b. In (b–f) error bars depict means ± SD (n = 4). shRNA, short hairpin RNA.

Figure 3

GL21.T aptamer inhibits cell migration and invasion. (a) Motility of U87MG and A549 cells was analyzed by Transwell Migration Assay in the presence of GL21.T or the unrelated aptamer for 24 hours toward 10% fetal bovine serum (FBS) or Gas6 as inducers of migration. (b) U87MG invasion through matrigel toward 10% FBS was carried out in the presence of GL21.T or the unrelated aptamer for 24 hours. In (a,b) the migrated or invaded cells, respectively, were stained with crystal violet and photographed. Representative photographs of at least three different experiments were shown. The results are expressed as percent of migrated or invaded cells in the presence of GL21.T with respect to cells treated with the unrelated aptamer. Vertical bars indicate the standard deviation values. (c) Serum-starved U87MG and A549 cells were either left untreated or treated for 3 hours with 200 nmol/l GL21.T or the unrelated aptamer and then stimulated with Gas6 in the presence of each aptamer. Rac1-GTP pull down assay was performed as described in Materials and Methods. The amount of total Rac1 was estimated by immunoblotting with anti Rac1 antibodies, as indicated. Values below the blots indicate signal levels relative to unrelated aptamer, arbitrarily set to 1 (labeled with asterisk). Quantitation was done as in Figure 1.

Figure 4

GL21.T aptamer inhibits spheroid formation. (a) U87MG spheroid diameter (left) and number (right) have been calculated following 10 days of treatment in the presence of GL21.T or the unrelated aptamer. (b) Spheroids average ~200 µm in diameter were seeded onto 24-well plates and allowed to adhere and migrate for 24 hours. Left, representative photographs of the spheroids before and after migration. Right, quantitation of U87MG cells migrated from the initial spheroids, error bars depict means ± SD (n = 10). Bar: 100 µm. (c) A549 cells were left untreated or treated for 48 hours with transforming growth factors β1 (TGFβ1) either in the absence or in the presence of GL21.T or the unrelated aptamer as reported in Materials and Methods. Left, cell viability was analyzed as in Figure 2. Right, proliferation was determined by [3H]-thymidine incorporation. (d,e) U87MG spheroids of ~200 µm in diameter were treated with GL21.T or the unrelated aptamer for 72 hours. (d) Spheroids were stained with anti-CD133 antibodies. (e) Cell viability was analyzed as in Figure 2. In (a–e), error bars depict means ± SD (n = 4).

Figure 5

GL21.T inhibits tumor growth. (a) Colony formation assay showing U87MG and A549 cells grown for 2 weeks in the presence of GL21.T or the unrelated aptamer. Representative photographs of at least three different experiments were shown. Bar: 100 µm. Colonies number of 15–20 random fields were counted and expressed as percent with respect to the unrelated aptamer-treated control. Vertical bars indicate the standard deviation values. (b) A549-luc xenografts were left growing for 30 days following implantation before aptamer injection. Mouse xenograft model bearing A549-luc cells tumors were injected intratumorally (left) or intravenously (right) with GL21.T or unrelated aptamer. Growth inhibition of tumors was measured as bioluminescence intensity (photon/sec) as indicated. Data shown are means ± SEM (n = 3 tumors). (c) Growth inhibition of tumors in a mouse xenograft model bearing A549 cells upon GL21.T treatment. Day 0 marks the first day of injection. Data are shown as means ± SEM (n = 8 tumors) (see Materials and Methods section for details). (d–g) Representative sections of tumors from unrelated aptamer or GL21.T-treated mouse were stained with (d,e) hematoxylin and eosin (H&E) and (f,g) Ki-67 antibody, as indicated. Magnification, ×200. (h) Three tumors per group selected randomly were excised, lysed, and the pooled or lysates were prepared from A549 cells treated for 24 hours with GL21.T or unrelated aptamer. Lysates were immunoblotted with anti-caspase-3, anti-PARP, and anti-α–tubulin antibodies, as indicated. Molecular weights of indicated proteins are reported.

Figure 6

GL21.T intravenous injection. (nu/nu) Mice-bearing MCF7-luc cells (left-hand side) and A549-luc (right-hand side) xenografts were injected intravenously either with 1,600 pmol of Alexa-labeled GL21.T or of unrelated aptamer. The aptamer amount in vivo was thus monitored by evaluating the intensity of fluorescence signal normalized for the tumor mass as determined by cell bioluminescence and measured at different times as indicated. (a) Typical image of one set of mice by IVIS camera at 180 minutes after injection. Arrows indicate the tumor regions (left-hand side MCF7-luc, right-hand side A549-luc). The corresponding fluorescent signal is reported for GL21.T (left mouse), unrelated aptamer (middle mouse), and sham control (right mouse). (b) The graph depicts the photon rate normalized by the bioluminescence signal (as measure of tumor volume) up to 110 minutes following GL21.T aptamer injection. The insert represents the signal measured from 140 to 1,620 minutes. (c) The same as in b for the mice injected with an unrelated aptamer sequence.