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A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent

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Science Translational Medicine  23 May 2018:
Vol. 10, Issue 442, eaah4807
DOI: 10.1126/scitranslmed.aah4807
  • Fig. 1 Unlike PLXNA4, NRP1 is necessary for SEMA3A-elicited EC collapse but not for SEMA3A inhibition of EC haptotaxis.

    (A) The C-terminal basic stretch of SEMA3A_WT binds the b1 domain of NRP1 with high-affinity (solid double arrow) NRP1, and this domain acts as a co-receptor that keeps SEMA3A_WT close to PLXNA4. SEMA3A_WT drives the activation of PLXNA4 guanosine triphosphatase (GTPase) activating protein (GAP) domain via a very low-affinity sema domain–sema domain interaction (dashed double arrow). The SEMA3A_ΔIg-b deletion mutant lacks the high-affinity NRP1-binding basic stretch and, after fusion with the mouse IgG1 constant fragment (Fc), is stably dimeric. Cyto, cytosolic domain; IPT, integrin-plexin-transcription factor domain; PM, plasma membrane. (B) In situ binding assays on NRP1 or green fluorescent protein (Mock)–transfected COS-7 cells evaluated the ability of AP-SEMA3A_WT and mutant AP-SEMA3A_ΔIg-b to bind to NRP1. Scale bar, 50 μm. (C) Real-time analysis of SEMA3A-elicited EC collapse as evaluated by an xCELLigence system. ECs were left to adhere on type I collagen (Coll I) for 2 hours. Different amounts (0.2 to 3.5 nM) of either SEMA3A_WT (green) or SEMA3A_ΔIg-b (blue) were then added or not to ECs, and cell spreading index was monitored over time. A representative experiment out of three is shown. For simplicity, data from the same experiment are plotted in two separate graphs: SEMA3A_WT (left graph) and SEMA3A_ΔIg-b (right graph). Each curve is the mean of three technical replicates ± SD. (D and E) Collapse of control (siCtl or shCtl), NRP1 (siNRP1)–silenced (D), or PLXNA4 (shPLXNA4)–silenced (E) ECs was analyzed over time upon addition of 3.5 nM SEMA3A_WT. A representative experiment out of six (D) or five (E) is depicted. Results are the mean of four technical replicates ± SD. (F) To label ligand-bound/active α5β1 integrins on the cell surface, living ECs were incubated for 10 min at 37°C with SNAKA51 mAb. ECs were then left to internalize SNAKA51-bound α5β1 integrins for 2 or 5 min either in the absence (control; gray) or in the presence of SEMA3A_WT (green) or SEMA3A_ΔIg-b (blue). After acid wash at 4°C, ECs were fixed and immunostained to visualize the early endosome marker early endosome antigen 1 (EEA1) (red in merge) and endocytosed SNAKA51–α5β1 integrin complexes (green). Representative single confocal z sections of ECs that were incubated for 5 min are shown. Mean fluorescence intensity (MFI) of SNAKA51 mAb accumulated in EEA1+ endosomes in six randomly chosen fields (≥18 cells per field) for each experimental point, from two independent experiments, was acquired at the same acquisition settings and quantified by ImageJ software. Scale bar, 20 μm. (G) Real-time analysis of haptotactic EC migration toward Coll I either in the absence (control, gray) or in the presence of 3.5 nM SEMA3A_WT (green) or SEMA3A_ΔIg-b (blue), assessed with an xCELLigence system. Results are the mean ± SEM of three independent experiments, with each experimental point performed in triplicate. (H to K) Haptotactic migration toward Coll I of control (shCtl or siCtl), PLXNA4 (shPLXNA4)–silenced (H and J), or NRP1 (siNRP1)–silenced (I and K) ECs was analyzed over time either in the absence (control, gray) or in the presence of 3.5 nM SEMA3A_WT (green) (H and I) or SEMA3A_ΔIg-b (blue) (J and K). For simplicity, data from the same experiments are plotted in two separate graphs: shCtl (H and J) or siCtl (I and K) (left graphs) and shPLXNA4 (H and J) or siNRP1 (I and K) (right graph). Results are the mean ± SEM of three (H and J), four (K), or five (I) independent experiments, with each experimental point having been performed in triplicate. Results were analyzed by a two-tailed heteroscedastic Student’s t test; ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 SEMA3A_ΔIg-b does not induce vascular permeability and slightly reduces tumor growth in RIP-Tag2.

    (A) Representative images showing Evans Blue extravasation from the mouse ear vasculature upon acute stimulation with 600 ng of recombinant SEMA3A_WT, SEMA3A_ΔIg-b, or VEGF-A 165, used as a positive control (n = 3 per group). Saline solution was used as a negative control (from here on, referred to as “control”). Scale bar, 1 mm. (B) Permeability was evaluated as the percentage of leaked blue/total area (mm2). SEMA3A_WT, but not SEMA3A_ΔIg-b, elicited vascular leakage. (C) Total tumor volume of RIP-Tag2 mice treated for 4 weeks with AAV8-SEMA3A_WT or AAV8-SEMA3A_ΔIg-b. Results are the mean ± SD (n = 5 per group). Results were analyzed by nonparametric two-tailed, unpaired Mann-Whitney U test; *P < 0.05 and **P < 0.01.

  • Fig. 3 In silico analyses reveal a central role for extrusion 1 in controlling SEMA3A-PLXNA4 molecular interactions.

    Representation of the overall electrostatic contacts at the interface of (A) SEMA6A-PLXNA2 and (B) SEMA3AA106K-PLXNA4 heterodimers, as determined by molecular dynamics simulations. SEMA and PLXNs are shown as blue and white cartoons, respectively. Residues involved in stable electrostatic interactions (as defined in table S1) are represented as stick and surface models and labeled with the one-letter code in the insets. For clarity, insets are rotated with respect to the corresponding overall representation. All the stick models are shown with carbon, nitrogen, oxygen, sulfur, and hydrogen atoms in cyan, blue, red, yellow, and white, respectively.

  • Fig. 4 The NRP1-independent SEMA3A_A106K_ΔIg-b mutant protein isoform binds to PLXNA4 with nanomolar affinity.

    (A) In situ assays of NRP1, PLXNA1 to PLXNA4, or green fluorescent protein (Mock)–transfected COS-7 cells assessed binding of AP-SEMA3A_WT, AP-SEMA3A_ΔIg-b, and AP-SEMA3A_A106K_ΔIg-b. Scale bar, 100 μm. (B to D) Binding curves (B and D) and Scatchard analysis (C) of PLXNA4 (B and C) or NRP1 (D) at different concentrations of ligands [(B) and (C), SEMA3A_A106K_ΔIg-b; (D), SEMA3A_WT], quantified by spectrometry of chromogenic conversion of AP substrate p-nitrophenyl phosphate. Abs405nm, absorbance at 405 nm.

  • Fig. 5 SEMA3A_A106K_ΔIg-b is more effective than SEMA3A_WT and SEMA3A_ΔIg-b in inhibiting directional migration and eliciting biochemical signaling in ECs.

    (A and B) Real-time analysis of haptotactic EC migration toward Coll I either in the absence (control, gray) or in the presence of 0.9 nM (A) or 3.5 nM (B) recombinant SEMA3A_WT (green), SEMA3A_ΔIg-b (blue), or SEMA3A_A106K_ΔIg-b protein (red) was performed with an xCELLigence system. Results are the mean ± SEM of three independent experiments, with each experimental point having been performed in triplicate. *SEMA3A_A106K_ΔIg-b versus SEMA3A_WT; #, SEMA3A_A106K_ΔIg-b versus SEMA3A_ΔIg-b. (C and D) Haptotactic migration toward Coll I of control (shCtl or siCtl), PLXNA4 (shPLXNA4)–silenced (C), or NRP1 (siNRP1)–silenced (D) ECs was analyzed over time either in the absence (control, gray) or in the presence (red) of 1.8 nM SEMA3A_A106K_ΔIg-b protein. For simplicity, data from the same experiments are plotted in two separate graphs: shCtl (C) or siCtl (D) (left graph) and shPLXNA4 (C) or siNRP1 (D) (right graph). Results are the mean ± SEM of three (C) or four (D) independent experiments, with each experimental point having been performed in triplicate. (E) Pull-down assay of active RAP1 GTP in ECs that were treated or not for 1 min with 0.02 nM SEMA3A_WT, SEMA3A_ΔIg-b, or SEMA3A_A106K_ΔIg-b protein. For each experimental point, total RAP1, detected in the input fraction, was used to calculate the normalized amount of active RAP1 (RAP1 GTP/total RAP1). Results are the mean ± SEM of three independent biological replicates, one of which is shown. (F) Western blot analysis of activated phospho-ERK1/2 in ECs that were treated or not for 15 min with 0.2 nM SEMA3A_WT, SEMA3A_ΔIg-b, or SEMA3A_A106K_ΔIg-b protein. Western blot analysis of total ERK1/2 was used to calculate the normalized amount of active ERK1/2 (pERK1/2/total ERK1/2). Results are the mean ± SEM of four independent biological replicates, one of which is shown. Statistical analysis results were analyzed by a two-tailed heteroscedastic Student’s t test; *P < 0.05, **/##P < 0.01, and ***/###P < 0.001.

  • Fig. 6 SEMA3A_A106K_ΔIg-b inhibits tumor growth and metastatic dissemination in RIP-Tag2 and mPDAC models.

    (A) Tumor volume of RIP-Tag2 mice treated with SEMA3A_A106K_ΔIg-b protein or control. (B) Confocal analysis of peripancreatic lymph node (LN) metastases, assessed by SV40 T-antigen (T-Ag) immunofluorescence (red); nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 μm. (C) Effect of treatment with SEMA3A_A106K_ΔIg-b protein or control on LN metastasis volume (n = 10 per group). (D) Graphs show tumor volume of mPDAC mice treated for 3 weeks (n = 5 per group) with AAV8-SEMA3A_A106K_ΔIg-b or AAV8-SEMA3A_WT or control. §Treated sample versus control. (E) Total area of liver metastasis measured and plotted as metastatic liver fraction; §treated sample versus control. (F and G) Tumor volume (F) and liver metastasis area (G) of mPDAC treated for 3 weeks (n = 5 per group) with AAV8-SEMA3A_A106K_ΔIg-b or with SEMA3A_A106K_ΔIg-b. (H) Survival of tumor-bearing mPDAC mice continuously treated with SEMA3A_A106K_ΔIg-b protein or with saline buffer and sacrificed for ethical reasons at a prespecified clinical end point (n = 14 mice per group). Results are the mean ± SD, and results were analyzed by nonparametric two-tailed, unpaired Mann-Whitney U test; *P < 0.05, **/§§P < 0.01, and ***P < 0.001; Kaplan-Meier survival curves were analyzed by Mantel-Cox test, **P < 0.01.

  • Fig. 7 SEMA3A_A106K_ΔIg-b normalizes the vasculature of RIP-Tag2 and mPDAC mice and sensitizes PDAC tumors to GEM.

    (A) Confocal analysis of Meca32 immunostaining evaluating vessel density in SEMA3A_A106K_ΔIg-b protein–treated RIP-Tag2 tumors and controls. (B) In RIP-Tag2 mice, blood vessel pericyte coverage after treatment with control or SEMA3A_A106K_ΔIg-b protein was evaluated by colocalization of Meca32 (green) and NG-2 (red). (C) Blood vessel perfusion was evaluated by lectin (green) colocalization with Meca32 (red) in RIP-Tag2 tumors. (D) Tumor hypoxia was assessed by pimonidazole adduct immunostaining in SEMA3A_A106K_ΔIg-b protein–treated RIP-Tag2 tumors and controls. (E) Confocal analysis revealed pericyte coverage in blood vessels of SEMA3A_A106K_ΔIg-b protein–treated PDAC mice and controls, as assessed by colocalization of Meca32 (green) and NG-2 (red). (F) Lectin (green) colocalized with Meca32 (red) showed vessel perfusion in mPDAC treated with SEMA3A_A106K_ΔIg-b protein and controls. (G) Tumor hypoxia was assessed in mPDAC after treatment with SEMA3A_A106K_ΔIg-b or control by immunostaining with carbonic anhydrase (CA9) antibody. Results are from five fields per mouse (n = 5 per treatment group). (H and I) mPDAC mice were treated for 3 weeks with the following: (i) SEMA3A_A106K_ΔIg-b protein (3 mg/kg, intraperitoneally, three times per week), (ii) GEM (50 mg/kg, intravenously, twice per week), (iii) SEMA3A_A106K_ΔIg-b protein + GEM, and (iv) control (saline solution) (n = 11 per group). (H) Analysis of tumor volume; the combination of the two drugs (SEMA3A_A106K_ΔIg-b + GEM) more efficiently reduced tumor burden (by 66%) compared with the vehicle-treatment group. §Treated sample versus control. (I) Analysis of the total area of liver metastasis; SEMA3A_A106K_ΔIg-b + GEM more effectively impaired the liver metastasis fraction compared to single drugs (by 90, 87, and 67% compared with controls, GEM, or SEMA3A_A106K_ΔIg-b alone, respectively). §Treated sample versus control. Results are the mean ± SD and were analyzed by nonparametric two-tailed, unpaired Mann-Whitney U test; *P < 0.05, **/§§P < 0.01, and ***/§§§P < 0.001. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/442/eaah4807/DC1

    Materials and Methods

    Fig. S1. Purification of Fc-tagged SEMA3A_ΔIg-b and SEMA3A_A106K_ΔIg-b recombinant proteins.

    Fig. S2. SEMA3A_ΔIg-b protein effect on tumor growth in RIP-Tag2 mice.

    Fig. S3. SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 interaction details.

    Fig. S4. Structural comparison of key residues involved in the charge complementarity of the extrusion region.

    Fig. S5. Root mean square deviation of the backbone atoms from the starting SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 structures as a function of time.

    Fig. S6. Root mean square fluctuation of α-carbon atoms of SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 structures.

    Fig. S7. B-factor analysis.

    Fig. S8. SEMA3A_A106K_ΔIg-b mutant protein isoform binding to PLXNA4 in the presence or the absence of NRP1.

    Fig S9. SEMA3B_A105K_ΔIg-b mutant protein isoform binding to PLXNA2.

    Fig. S10. Testing SEMA3A_A106K_ΔIg-b mutant isoform in EC collapse assays.

    Fig. S11. NRP1-independent SEMA3A_A106K_ΔIg-b biochemical signaling in ECs.

    Fig. S12 Comparing SEMA3A_ΔIg-b and SEMA3A_A106K_ΔIg-b effects on tumor growth in RIP-Tag2 and mPDAC.

    Fig. S13. Monitoring the effect of SEMA3A_A106K_ΔIg-b protein on mPDAC tumor growth over time.

    Fig. S14. Toxicology analysis of SEMA3A_A106K_ΔIg-b protein–treated mice.

    Fig. S15. Rotarod motor performance analysis of SEMA3A_A106K_ΔIg-b protein–treated mice.

    Fig. S16. Effect of SEMA3A_A106K_ΔIg-b protein on laser-induced CNV of the mouse retina.

    Table S1. Analysis of the electrostatic heterodimer interfaces.

    Table S2. Biochemical analysis of renal and hepatic function parameters.

    Table S3. Hematological profiling of all the treatment groups compared with the controls.

    Table S4. Raw data (provided as an Excel file).

    References (7289)

  • Supplementary Material for:

    A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent

    Noemi Gioelli, Federica Maione, Chiara Camillo, Michela Ghitti, Donatella Valdembri, Noemi Morello, Marie Darche, Lorena Zentilin, Gabriella Cagnoni, Yaqi Qiu, Mauro Giacca, Maurizio Giustetto, Michel Paques, Ilaria Cascone, Giovanna Musco, Luca Tamagnone, Enrico Giraudo,* Guido Serini*

    *Corresponding author. Email: guido.serini{at}ircc.it (G.S.); enrico.giraudo{at}ircc.it (E.G.)

    Published 23 May 2018, Sci. Transl. Med. 10, eaah4807 (2018)
    DOI: 10.1126/scitranslmed.aah4807

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Purification of Fc-tagged SEMA3A_ΔIg-b and SEMA3A_A106K_ΔIg-b recombinant proteins.
    • Fig. S2. SEMA3A_ΔIg-b protein effect on tumor growth in RIP-Tag2 mice.
    • Fig. S3. SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 interaction details.
    • Fig. S4. Structural comparison of key residues involved in the charge complementarity of the extrusion region.
    • Fig. S5. Root mean square deviation of the backbone atoms from the starting SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 structures as a function of time.
    • Fig. S6. Root mean square fluctuation of α-carbon atoms of SEMA6A-PLXNA2 and SEMA3AA106K-PLXNA4 structures.
    • Fig. S7. B-factor analysis.
    • Fig. S8. SEMA3A_A106K_ΔIg-b mutant protein isoform binding to PLXNA4 in the presence or the absence of NRP1.
    • Fig S9. SEMA3B_A105K_ΔIg-b mutant protein isoform binding to PLXNA2.
    • Fig. S10. Testing SEMA3A_A106K_ΔIg-b mutant isoform in EC collapse assays.
    • Fig. S11. NRP1-independent SEMA3A_A106K_ΔIg-b biochemical signaling in ECs.
    • Fig. S12. Comparing SEMA3A_ΔIg-b and SEMA3A_A106K_ΔIg-b effects on tumor growth in RIP-Tag2 and mPDAC.
    • Fig. S13. Monitoring the effect of SEMA3A_A106K_ΔIg-b protein on mPDAC tumor growth over time.
    • Fig. S14. Toxicology analysis of SEMA3A_A106K_ΔIg-b protein–treated mice.
    • Fig. S15. Rotarod motor performance analysis of SEMA3A_A106K_ΔIg-b protein–treated mice.
    • Fig. S16. Effect of SEMA3A_A106K_ΔIg-b protein on laser-induced CNV of the mouse retina.
    • Table S1. Analysis of the electrostatic heterodimer interfaces.
    • Table S2. Biochemical analysis of renal and hepatic function parameters.
    • Table S3. Hematological profiling of all the treatment groups compared with the controls.
    • References (7289)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Raw data (provided as an Excel file).

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