Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.
RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC) with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA). Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM) were selected which showed significant inhibition (p<0.001) of gene (RT-qPCR) and protein (Western blot) expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3) but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1) showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001) inhibitory effects on RP3 cell viability (MTT assay) and on invasion/migration (IncuCyte scratch test) with blockage of cell cycle at G0/G1 phase (flow cytometry) and induced apoptosis by caspase-3 and PARP1 cleavage (WB). After intravenous injection in nude mice, respective squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC3 significantly (p<0.001) reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67). Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.
Université Paris-Sud 11, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; CNRS, Villejuif, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France.
Hafiz Muhammad
HM
Ali
Université Paris-Sud 11, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; CNRS, Villejuif, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France.
Giorgia
G
Urbinati
Institut Galien, UMR CNRS 8612, Université Paris-Sud 11, Faculté de pharmacie, Châtenay-Malabry, France.
Hubert
H
Chapuis
Institut Galien, UMR CNRS 8612, Université Paris-Sud 11, Faculté de pharmacie, Châtenay-Malabry, France.
Didier
D
Desmaele
Université Paris-Sud 11, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; CNRS, Villejuif, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France.
Jean-Rémi
JR
Bertrand
Institut Galien, UMR CNRS 8612, Université Paris-Sud 11, Faculté de pharmacie, Châtenay-Malabry, France.
Patrick
P
Couvreur
Université Paris-Sud 11, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; CNRS, Villejuif, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France; Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France.
Liliane
L
Massaad-Massade
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000737:chemistry; D002277:Carcinoma / Q000188:drug therapy* / Q000473:pathology; D002291:Carcinoma, Papillary; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D009368:Neoplasm Transplantation; D056918:Nuclear Receptor Coactivators / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000037:antagonists & inhibitors* / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000494:pharmacology; D013185:Squalene / Q000378:metabolism*; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000473:pathology
D000970:Antineoplastic Agents; D056918:Nuclear Receptor Coactivators; D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; D013185:Squalene; D051096:Proto-Oncogene Proteins c-ret