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. 2014 Apr;22(4):828-41.
doi: 10.1038/mt.2013.300. Epub 2014 Jan 2.

Inhibition of receptor signaling and of glioblastoma-derived tumor growth by a novel PDGFRβ aptamer

Simona Camorani  1 , Carla L Esposito  2 , Anna Rienzo  2 , Silvia Catuogno  2 , Margherita Iaboni  3 , Gerolama Condorelli  1 , Vittorio de Franciscis  2 , Laura Cerchia  2
Affiliations

Inhibition of receptor signaling and of glioblastoma-derived tumor growth by a novel PDGFRβ aptamer

Simona Camorani et al. Mol Ther. 2014 Apr.

Abstract

Platelet-derived growth factor receptor β (PDGFRβ) is a cell-surface tyrosine kinase receptor implicated in several cellular processes including proliferation, migration, and angiogenesis. It represents a compelling therapeutic target in many human tumors, including glioma. A number of tyrosine kinase inhibitors under development as antitumor agents have been found to inhibit PDGFRβ. However, they are not selective as they present multiple tyrosine kinase targets. Here, we report a novel PDGFRβ-specific antagonist represented by a nuclease-resistant RNA-aptamer, named Gint4.T. This aptamer is able to specifically bind to the human PDGFRβ ectodomain (Kd: 9.6 nmol/l) causing a strong inhibition of ligand-dependent receptor activation and of downstream signaling in cell lines and primary cultures of human glioblastoma cells. Moreover, Gint4.T aptamer drastically inhibits cell migration and proliferation, induces differentiation, and blocks tumor growth in vivo. In addition, Gint4.T aptamer prevents PDGFRβ heterodimerization with and resultant transactivation of epidermal growth factor receptor. As a result, the combination of Gint4.T and an epidermal growth factor receptor-targeted aptamer is better at slowing tumor growth than either single aptamer alone. These findings reveal Gint4.T as a PDGFRβ-drug candidate with translational potential.

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Figures

Figure 1
Figure 1
Gint4.T aptamer specifically binds to human PDGFRβ and rapidly internalizes into GBM cells. (a) Secondary structure of Gint4.T predicted by using DNAsis software. (b) Binding isotherm for Gint4.T: EC-PDGFRβ complex. (c) Binding of 100 nmol/l radiolabeled Gint4.T, prior incubated with 200 nmol/l EC-PDGFRα or EC-PDGFRβ for 15 minutes at 37 °C, to U87MG cells. In b, c, the results are expressed relative to the background binding detected with the unrelated aptamer, used as a negative control. (d–k) Following 10-minutes FAM-Gint4.T treatment, U87MG, U87MG/shRNAPDGFRβ (U87MG cells following 72 hour-transfection with a specific PDGFRβ short hairpin RNA) or A549 cells were stained with anti-PDGFRβ antibodies, visualized by confocal microscopy and photographed. (l) Internalization rate of radiolabeled Gint4.T and unrelated aptamer into U87MG cells. Results are expressed as percentage of internalized RNA relative to total bound aptamer. In b, c, l error bars depict mean ± SD (n = 3). (m–t) Following treatment with FAM-Gint4.T for the indicated times, U87MG cells were stained with anti-EEA1 (m–p) or LAMP1 (q–t) antibodies, visualized by confocal microscopy and photographed. (d–k, m–t) All digital images were captured at the same setting to allow direct comparison of staining patterns. Scale bars = 10 µm. The Manders' coefficients for the amount of co-localization were: M1, 0.975 and M2, 0.908 (g); M1, 0.620 and M2, 0.615 (p); M1, 0.980 and M2, 0.972 (t). EEA1, early endosome antigen 1; GBM, glioblastoma; LAMP1, lysosomal-associated membrane protein 1; PDGFRβ, platelet-derived growth factor receptor β.
Figure 2
Figure 2
Gint4.T inhibits PDGF-BB-dependent PDGFRβ activation. (a–c) Serum-starved T98G, U87MG, or primary glioma cells from two patients (PG-G and VS-GB) were either left untreated or stimulated with PDGF-BB in the presence of Gint4.T or the unrelated aptamer (used as a negative control), as indicated. Cell lysates were immunoblotted with anti-pPDGFRβ, anti-PDGFRβ, anti-pERK, anti-pAkt. Filters were stripped and reprobed with anti-Erk and anti-Akt antibodies, as indicated. Values below the blots indicate signal levels relative to PDGF-BB stimulated cells in the absence (a) or in the presence (b, c) of unrelated aptamer, arbitrarily set to 1 (labeled with asterisk). (a–c) Equal loading was confirmed by immunoblot with anti-α-tubulin antibody. Molecular weights of indicated proteins are reported. PDGFRβ, platelet-derived growth factor receptor β.
Figure 3
Figure 3
Gint4.T inhibits GBM cell migration. (a) Motility of T98G and U87MG cells was analyzed by Transwell Migration Assay in the presence of Gint4.T or the unrelated aptamer, used as a negative control, for 24 hours toward 10% FBS or PDGF-BB (50 ng/ml) as inducers of migration. The migrated cells 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 cells in the presence of Gint4.T with respect to cells treated with the unrelated aptamer. (b) Confluent monolayers of T98G and U87MG cells were subjected to scratch assays and mock-treated or treated with Gint4.T or the unrelated aptamer for 24 and 48 hours. Phase-contrast microscopy images were taken at the indicated time and the extent of wound closure was calculated (magnification 4×). (a, b) ***P < 0.0001 relative to unrelated (n = 3). Error bars depict means ± SD. FBS, fetal bovine serum; PDGF, platelet-derived growth factor.
Figure 4
Figure 4
Gint4.T inhibits GBM cell survival and proliferation. T98G (a) and U87MG (b) cells were mock-treated or treated for 24 hours with increasing amounts of Gint4.T, CL4 or the unrelated aptamer as a negative control, or with 200 nmol/l final concentration of each aptamer for the indicated incubation times (inserts). Cell viability was analyzed and expressed as percent of viable treated cells with respect to mock-treated cells. (a, b) P values for Gint4.T and CL4 relative to unrelated are: ***P < 0.0001; **P < 0.005; *P < 0.05 (n = 6). Error bars depict means ± SD. T98G (c) and U87MG (d) cells were treated for 72 hours with Gint4.T or the unrelated aptamer, as indicated. Cell-cycle profile were determined by BrdU incorporation and PI staining. Percentages of cells in each cycle phase are indicated. BrdU, anti-5-bromodeoxyuridine; PI, propidium iodide.
Figure 5
Figure 5
Gint4.T induces GBM cell differentiation. T98G (a) and U87MG (b) cells were either mock-treated or treated with Gint4.T or the unrelated aptamer, used as a negative control, by renewing the aptamer treatment each 24 hours and the cell number was counted at the indicated time points. In (a), at day 4 of Gint4.T treatment, the aptamer was removed from the culture medium (the arrow) and incubation prolonged (dashed line). (a,b) Growth curves represent the average of three independent experiments. ***P < 0.0001; **P < 0.005; *P < 0.05 relative to unrelated (n = 6). Error bars represent mean ± SD. (c) T98G and U87MG cells were treated for 6 days with Gint4.T or the unrelated aptamer and photographed by phase-contrast microscopy (magnification 4×). (d) Cells were treated as in (c) or with 1 µmol/l ATRA and GFAP mRNA levels were analyzed by RT-PCR. ATRA, all-trans retinoic acid; GFAP, glial fibrillary acidic protein.
Figure 6
Figure 6
Gint4.T prevents PDGFRβ-mediated EGFR transactivation. (a, b) Lysates from T98G cells, following transfection with shRNAPDGFRβ or shRNActrl (a) or treated for 6-hours with Gint4.T or the unrelated aptamer as a negative control (b), were immunoblotted with anti-pPDGFRβ, anti-PDGFRβ, anti-pEGFR, anti-EGFR antibodies, as indicated. (c) Serum-starved U87MG, T98G, and A431 cells were either left untreated or stimulated with PDGF-BB or EGF and cell lysates were immunoblotted with anti-pEGFR, anti-EGFR antibodies, as indicated. (d, e) Serum-starved T98G cells were either left untreated or stimulated with PDGF-BB in the absence or in the presence of 200 nmol/l Gint4.T, 200 nmol/l CL4, 200 nmol/l Gint4.T plus 200 nmol/l CL4, or 400 nmol/l unrelated aptamer, as indicated. Cell lysates were either immunoblotted with anti-pPDGFRβ, anti-PDGFRβ, anti-pEGFR, anti-EGFR antibodies (d) or immunoprecipitated with anti-PDGFRβ antibody and immunoblotted with anti-pEGFR and anti-EGFR antibodies (e). (a–e) Values below the blot indicate signal levels relative to each control, arbitrarily set to 1 (labeled with asterisk). (a–d) Equal loading was confirmed by immunoblot with anti-α-tubulin antibody. Molecular weights of indicated proteins are reported. (f, g) T98G and U87MG cells were mock-treated or treated for 72 hours with 200 nmol/l Gint4.T, 200 nmol/l CL4, and (f) 10 µmol/l Imatinib (indicated as Ima), 5 µmol/l Gefitinib (indicated as Gef), 1 µmol/l Cetuximab (indicated as Cet), or (g) 100 µmol/l and 400 µmol/l TMZ, as single agents or in combination, as indicated and cell viability was analyzed. (f) As a negative control, cells were treated with the unrelated aptamer at a concentration of 400 nmol/l. ***P < 0.0001; **P < 0.005; *P < 0.05 relative to mock-treated (n = 6). ###P < 0.0001. Error bars depict means ± SD. EGFR, epidermal growth factor receptor; PDGFRβ, platelet-derived growth factor receptor β; TMZ, temozolomide.
Figure 7
Figure 7
Gint4.T cell specificity in vivo and inhibition of tumor growth. (a, b) Mice bearing MCF7-luc (right-flank) and U87MG-luc (left-flank) xenografts (tumor mean volume: 60 mm3) were injected intravenously either with Alexa-labeled Gint4.T or the unrelated aptamer, used as a negative control. (a) Aptamer amount was monitored by evaluating the intensity of fluorescent signal normalized for the bioluminescence and measured at the indicated times. Example shows fluorescence signal in one representative animal from each treatment group at 120 minutes after injection. The circles indicate where the tumors are located. (b) Tumor growth inhibition was measured as bioluminescence intensity (photons/second). ***P < 0.0001 (n = 5). (c) Mice bearing U87MG-luc xenografts (tumor mean volume, 150 mm3) were injected intravenously with Gint4.T, CL4, Gint4.T plus CL4, unrelated aptamer or PBS (vehicle) at day 0, 3, 5, and 7. Tumor volumes were measured by bioluminescence and calipers (insert) and experimental raw data (expressed as fold increase) were interpolated with no curve fitting or regression analysis. **P < 0.005; *P < 0.05 relative to vehicle (n = 5). In (b, c) day 0 marks the start of treatments. (d) Immunoblot with anti-pPDGFRβ, anti-PDGFRβ, anti-pEGFR, anti-EGFR, and anti-α-tubulin antibodies of pooled lysates from recovered tumors. Values are expressed as relative to vehicle, arbitrarily set to 1 (labeled with asterisk). (e) Representative sections of tumors from each groups were stained with H&E and Ki-67 antibody, and Ki-67 proliferation index was calculated. ***P < 0.0001; **P < 0.005 relative to vehicle (n = 5). ###P < 0.0001. Scale bars are indicated. (f) Immunoblot with anti-caspase-3 and anti-α-tubulin antibodies of pooled lysates from recovered tumors. (g) P56 and OAS1 mRNAs expression relative to control arbitrarily set to 1. PBS and Poly (I:C)-treated mice were used as a negative and positive control, respectively. In (a–c, e, g) error bars depict means ± SD. EGFR, epidermal growth factor receptor; PDGFRβ, platelet-derived growth factor receptor β.
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