Anti-cancer effect of GV1001 for prostate cancer: function as a ligand of GnRHR

in Endocrine-Related Cancer
Correspondence should be addressed to K W Kang: kwkang@snu.ac.kr
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GV1001, a 16-amino acid fragment of the human telomerase reverse transcriptase catalytic subunit (hTERT), has been developed as an injectable formulation of cancer vaccine. Here, we revealed for the first time that GV1001 is a novel ligand for gonadotropin-releasing hormone receptor (GnRHR). The docking prediction for GV1001 against GnRHR showed high binding affinity. Binding of GV1001 to GnRHR stimulated the Gαs-coupled cAMP signaling pathway and antagonized Gαq-coupled Ca2+ release by leuprolide acetate (LA), a GnRHR agonist. Repeated injection of GV1001 attenuated both serum testosterone level and seminal vesicle weight via desensitization of hypothalamic–pituitary–gonadal (HPG) axis. We then tested whether GV1001 has an inhibitory effect on tumor growth of LNCaP cells, androgen receptor–positive human prostate cancer (PCa) cells. GV1001 significantly inhibited tumor growth and induced apoptosis in LNCaP-implanted xenografts. Interestingly, mRNA expressions of matrix metalloproteinase 2 and matrix metalloproteinase 9 were suppressed by GV1001, but not by LA. Moreover, GV1001 significantly inhibited the proliferation and migration of PCa cells and induced apoptosis in a concentration-dependent manner. Our findings suggest that GV1001 functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway, with anti-proliferative and anti-migratory effects on human PCa.

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  • Supplementary materials and methods
  • Supplementary Fig. 1. MALDI MS protein mass spectrum of biotinylated GV1001.

 

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    Binding of GV1001 to GnRHR. (A) Peptide structures of GV1001 and GnRH. Red-colored residues are overlapped with GnRH. (B) Peptide superposition analysis showing structural comparison between GV1001 and GnRH analogs, leuprolide acetate (LA) and cetrorelix acetate (CA). (C) Western blot analyses of GnRHR in HEK293-pcDNA3.1(+) and HEK293-GnRHR cells. Expression levels of the ß-actin proteins were determined as loading controls. Densitometric analyses were performed by three independent experiments and data represent means ± s.d., *P < 0.05 vs pcDNA3.1(+)-transfected control. (D) GV1001 binding with plasma membrane fraction. Fluorescence intensity was quantified to measure FITC-labeled GV1001 binding to membrane fraction from GnRHR-overexpressing or mock-transfected HEK293 cells. Data represent means ± s.d. (n = 3, *P < 0.05 vs pcDNA3.1(+)-transfected control. (E) GV1001 binding to GnRHR. Streptavidin-biotin binding assays were performed to confirm the direct interaction between GV1001 and GnRHR. Total cell lysates (300 μg) from pcDNA3.1(+) and HEK293-GnRHR cells were incubated with or without 4 nmol biotinylated GV1001 for 8 h. The results were confirmed by multiple experiments. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0454.

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    Docking studies of GV1001 to GnRHR. (A) Overall architecture of the first hGnRHR homology model from Bovine Rhodopsin and GV1001 bound ECL of hGnRHR. Atoms of GV1001 are shown as spheres. (B) Residues of hGnRHR within 5 Å of GV1001 in the first hGnRHR homology model (Left) GV1001: yellow stick, green dashed lines: hydrogen-bond interaction, pink dashed lines: hydrophobic interaction. (Right) Molecular surface of hGnRHR: gray solid, GV1001: ball & stick with colors assigned by Kyte–Doolittle hydrophobic scale, yellow dashed lines: hydrogen-bond interaction. (C) (Left) Superimposed structures of the second hGnRHR homology model and APJR-AMG3054-bound Human Apelin Receptor (PDB:5VBL) form a hydrogen-bond network with conserved amino acids, (Right) Overall architecture of the second hGnRHR homology model and GV1001 bound ECL of hGnRHR. Atoms of GV1001 are shown as spheres. (D) Residues of hGnRHR within 5 Å of GV1001 in the second hGnRHR homology model (Left) GV1001: yellow stick, green dashed lines: hydrogen-bond interaction, pink dashed lines: hydrophobic interaction. (Right) Molecular surface of hGnRHR: gray solid, GV1001: ball & stick with colors assigned by Kyte–Doolittle hydrophobic scale, yellow dashed lines: hydrogen-bond interaction.

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    Selective activation of Gαs/cAMP pathway by GV1001. (A and B) Intracellular calcium increase by GV1001 or LA. (A) LNCaP cells were incubated with LA (10 and 100 nM) or GV1001 (0.1–10 μM) for 60 min. Data represent means ± s.d. (n = 12–29, *P < 0.05, **P < 0.01 vs vehicle-treated control). (B) HEK293-pcDNA3.1(+) and HEK293-GnRHR cells were incubated with LA (100 nM) or GV1001 (0.01–10 μM) for 60 min. Data represent means ± s.d. (n = 28–34, **P < 0.01 vs vehicle-treated control). (C) GV1001-mediated functional antagonism on intracellular calcium increase by LA. LNCap cells were exposed to 100 nM LA for 60 min in the presence or absence of GV1001 (0.1–10 μM) or 100 nM CA. Data represent means ± s.d. (n = 43–52, **P < 0.01 vs LA-treated group). Effects of LA (D) and GV1001 (E) on CRE-luciferase activity in HEK293-pcDNA3.1(+) and HEK293-GnRHR cells. HEK293-pcDNA3.1(+) and HEK293-GnRHR cells were transiently transfected with CRE-luciferase plasmid (1 μg/mL) and the cells were exposed to LA (0.1–100 nM) or GV1001 (0.01–10 μM) for 24 h. Luciferase activity was determined to assess cAMP responsiveness. Data represent means ± s.d. (n = 4, **P < 0.01 vs vehicle-treated control). (F) Silencing of GnRHR by siRNA. LNCaP cells were transfected with nonspecific (Control) or GnRHR siRNA. GnRHR protein expression in both the cell types was determined by immunoblotting. Densitometric analysis was performed by three independent experiments and data represent means ± s.d., *P < 0.05 vs control siRNA. (G) LA-mediated functional antagonism on GV1001-induced increase in CRE-luciferase activity. LNCaP cells were transiently co-transfected with CRE-luciferase plasmid (1 μg/mL), siRNA (75 nmol/L) and phRL-SV (hRenilla) (10 ng/mL) and then the cells were exposed to GV1001 (10 μM), LA (100 nM) or forskolin (10 μM) for 24 h. Data represent means ± s.d. (n = 4, **P < 0.01 vs vehicle-treated control; #P < 0.05 vs GV1001-treated group). (H and I) Arg10- and Arg12-dependent activation of Gαs/cAMP pathway by GV1001. LNCaP cells (H) or HEK293-pcDNA3.1(+) and HEK293-GnRHR cells (I) were transiently transfected with CRE-luciferase plasmid (1 μg/mL) and the cells were exposed to 10 μM GV1001 or Arg10-, Arg12- and both of them substituted GV1001 peptides (R10A; R12A; R10A, R12A, 0.1–10 μM) for 24 h. Luciferase activity was determined to assess cAMP responsiveness. Data represent means ± s.d. (n = 4, *P < 0.05 vs vehicle-treated control).

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    In vivo anti-cancer effect of GV1001. (A) Testosterone level in the serum of vehicle or GV1001 (10 mg/kg)-injected BALB/c mice was measured by ELISA. Data represent means ± s.d. (n = 10, *P < 0.05, **P < 0.01 vs vehicle-treated control group). (B) Whole mounts of seminal vesicles collected from BALB/c mice after 7 days treatment of either vehicle or GV1001 (10 mg/kg). Quantitative analysis of seminal vesicle weights (% body weight) was presented as mean ± s.d. (n = 10, *P < 0.05 vs vehicle-treated control group). (C) Tumor growth of LNCaP cells-implanted xenografts. Left panel, representative images of tumor-bearing mice injected with vehicle, GV1001 (10 mg/kg) or LA (0.1 mg/kg) on 13 days after treatments; Right panel, time-dependent changes in tumor volume and body weight. Data represent means ± s.d. (n = 5, *P < 0.05 vs vehicle-treated control group). (D) TUNEL assay of the xenograft tumor sections from the mice treated with vehicle, GV1001 (10 mg/kg) or LA (0.1 mg/kg). TUNEL-positive cells were stained with brown color. TUNEL-positive area was calculated as described in material and methods section. Data represent means ± s.d. (n = 5, *P < 0.05, **P < 0.01 vs vehicle-treated control group). (E) Ki-67 immunohistochemical staining of the xenograft tumor sections. Data represent means ± s.d. (n = 5, **P < 0.01 vs vehicle-treated control group). (F) mRNA levels of MMP2 and MMP9. RT-qPCR was performed in xenograft tumor tissues. The expression levels were normalized by ß-actin mRNA. Data represent means ± s.d. (n = 5, *P < 0.05, **P < 0.01 vs vehicle-treated control group). (G) Expression levels of GnRHR in LNCaP, PC-3 cells and N87 cells. (H) Tumor growth of N87 cells-implanted xenografts. 30 mg/kg GV1001 showed no inhibitory effect on N87-tumor growth. 50 mg/kg 5-FU was intraperitoneally injected as positive control. Data represent means ± s.d. (n = 6, **P < 0.01 vs vehicle-treated control group). A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0454.

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    Effects of GV1001 on cell proliferation and migration in PCa cells. (A) Penetration of GV1001 in LNCaP cells. LNCaP cells were treated with 10 μM FITC-labeled GV1001. The peptide distribution was analyzed by confocal microscopy. (B and C) Effects of GV1001 (B) and LA (C) on cell proliferation of LNCaP cells. LNCaP cells were plated in 96-well plate and cell proliferation was determined by MTT assays after 96 h treatment with GV1001 (0.01–10 μM) or LA (1–100 nM). Data represent means ± s.d. (n = 6, *P < 0.05, **P < 0.01 vs vehicle-treated control). (D) GnRHR-mediated inhibition of LNCaP cell proliferation by GV1001. Control siRNA-transfected and GnRHR siRNA-transfected LNCaP cells were treated with GV1001 (0.1–10 μM) for 96 h and cell proliferation was determined by IncuCyte ZOOM live-cell analysis system. Data represent means ± s.d. (n = 6, *P < 0.05 vs vehicle-treated control). (E and F) Effects of GV1001 (E) and LA (F) on cell migration. Transwell migration assays were performed in LNCaP cells 18 h after treatment with GV1001 (1 and 10 μM) or LA (1, 10 and 100 nM). Representative pictures of migrated cells were shown (left). The relative cell numbers of migrated cells were counted (right). Data represent means ± s.d. (n = 12, **P < 0.01 vs vehicle-treated control). (G) Expression levels of GnRHR and AR in both AR-positive LNCaP cells and AR-negative PC-3 cells. Expression level of the ß-actin protein was used as a loading control. The mRNA expression levels were normalized by GAPDH mRNA. Data represent means ± s.d. from three independent experiments. *P < 0.05 vs LNCaP cells. (H) Effect of GV1001 on cell proliferation of PC-3 cells. PC-3 cells were plated in 96-well plate and cell proliferation was determined by MTT assay after 96 h treatment with GV1001 (0.01–10 μM). Data represent means ± s.d. (n = 6, **P < 0.01 vs vehicle-treated control. (I) Effect of GV1001 on cell migration of PC-3 cells. Transwell migration assays were performed in PC-3 cells 18 h after treatment with GV1001 (1 and 10 μM). Representative pictures of migrated cells were shown (left). The relative cell numbers of migrated cells were counted (right). Data represent means ± s.d. (n = 12–15, *P < 0.05, **P < 0.01 vs vehicle-treated control). A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0454.

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    GnRHR-dependent apoptosis induction by GV1001. (A) Concentration-dependent effect of GV1001 on caspase-3/7 activity in LNCaP cells. LNCaP cells were exposed to GV1001 (0.1, 1, and 10 μM) for 72 h and caspase-3/7 activity was determined by IncuCyte zoom system. Data represent means ± s.d. (n = 6, **P < 0.01 vs vehicle-treated control). (B) Comparison of apoptosis activity in GV1001-, CA- and LA-treated LNCaP cells. Caspase3/7 activity was determined in LNCaP cells exposed to 0.1 μM each compound for 72 h. Data represent means ± s.d. (n = 6, **P < 0.01 vs vehicle-treated control). (C) Determination of apoptosis marker proteins in LNCaP cells. LNCaP cells were incubated with or without GV1001 (0.1–10 μM) for 72 h and the total cell lysates were subjected to immunoblottings of PARP1, cleaved PARP1 (cl-PARP1), cleaved caspase-3 (cl-casp3) and Bcl-2. ß-actin was used as loading controls. Data represent means ± s.d. from three independent experiments. *P < 0.05, **P < 0.01 vs vehicle-treated control. (D) Concentration-dependent effect of GV1001 on caspase-3/7 activity in PC-3 cells. Data represent means ± s.d. (n = 6, **P < 0.01 vs vehicle-treated control). (E) Determination of cl-PARP1 in PC-3 cells. Data represent means ± s.d. from three independent experiments. *P < 0.05, **P < 0.01 vs vehicle-treated control. (F) Effect of GnRHR siRNA on caspase-3/7 activity. Control siRNA or GnRHR siRNA-transfected LNCaP cells were exposed to 10 μM GV1001 for 72 h and caspase-3/7 activity was determined by IncuCyte ZOOM system. Data represent means ± s.d. (n = 3, **P < 0.01 vs vehicle-treated control; ##P < 0.01 vs control siRNA-transfected group). (G) Protein levels of GnRHR in LNCaP-Control sgR/Cas9 and LNCaP-GnRHR sgR/Cas9 cells. Data represent means ± s.d. (n = 4, *P < 0.05 vs LNCaP-Control sgR/Cas9 cells). (H) Tumor growth of LNCaP-Control sgR/Cas9- or LNCaP-GnRHR sgR/Cas9-implanted xenografts. Left panel, representative images of tumor-bearing mice injected with or without GV1001 (10 mg/kg) for 13 days; Right panel, time-dependent changes in tumor volume. Data represent means ± s.d. (n = 5, **P < 0.01 vs vehicle-treated control group). A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0454.

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