title: Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells.
abstract: Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells-a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells-to evaluate its involvement in promoting proliferation, invasiveness, resistance to apoptosis and stemness. By RT-PCR, methylation-specific PCR, fluorescent in situ hybridization, DNA sequence, western blot and immunofluorescence analyses, we have shown that-in comparison with parental MG63 cells where TP53 gene is hypermethylated, rearranged and in single copy-in 3AB-OS cells, TP53 is unmethylated, rearranged and in multiple copies, and mutp53 (p53-R248W/P72R) is post-translationally modified and with nuclear localization. p53-R248W/P72R-knockdown by short-interfering RNA reduced the growth and replication rate of 3AB-OS cells, markedly increasing cell cycle inhibitor levels and sensitized 3AB-OS cells to TRAIL-induced apoptosis by DR5 up-regulation; moreover, it strongly decreased the levels of stemness and invasiveness genes. We have also found that the ectopic expression of p53-R248W/P72R in MG63 cells promoted cancer stem-like features, as high proliferation rate, sphere formation, clonogenic growth, high migration and invasive ability; furthermore, it strongly increased the levels of stemness proteins. Overall, the findings suggest the involvement of p53-R248W/P72R at the origin of the aberrant characters of the 3AB-OS cells with the hypothesis that its GOF can be at the root of the dedifferentiation of MG63 cells into CSCs.
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Di Fiore
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Marcatti
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Drago-Ferrante
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: D'Anneo
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Giuliano
authors.affiliation: Laboratory of Biochemistry, Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Carlisi
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: De Blasio
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy; CEINGE-Advanced Biotechnology s.c.a.r.l., Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Querques
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II," Naples, Italy; CEINGE-Advanced Biotechnology s.c.a.r.l., Naples, Italy.
authors.forename: Lucio
authors.initials: L
authors.lastname: Pastore
authors.affiliation: Institute for Cancer Research and Molecular Medicine and Center of Biotechnology, College of Science and Biotechnology, Temple University, Philadelphia, PA, USA.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Tesoriere
authors.affiliation: Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy; Institute for Cancer Research and Molecular Medicine and Center of Biotechnology, College of Science and Biotechnology, Temple University, Philadelphia, PA, USA. Electronic address: renza.vento@unipa.it.
authors.forename: Renza
authors.initials: R
authors.lastname: Vento
mesh_terms: D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism; D054337:Cell Dedifferentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D012516:Osteosarcoma / Q000235:genetics* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D053218:Receptors, Death Domain / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D014410:Tumor Stem Cell Assay; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D019253:Proto-Oncogene Proteins c-bcl-2; D053218:Receptors, Death Domain; D053221:TNF-Related Apoptosis-Inducing Ligand; D016159:Tumor Suppressor Protein p53
keywords: 3AB-OS cells; Cancer cell dedifferentiation; Cancer stem cells; Human osteosarcoma; Mutant p53 gain of function