TY - JOUR
T1 - Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells
AU - Di Fiore, Riccardo
AU - Marcatti, Michela
AU - Drago-Ferrante, Rosa
AU - D'Anneo, Antonella
AU - Giuliano, Michela
AU - Carlisi, Daniela
AU - De Blasio, Anna
AU - Querques, Francesca
AU - Pastore, Lucio
AU - Tesoriere, Giovanni
AU - Vento, Renza
N1 - Funding Information:
This work was partially funded by the European Regional Development Fund, European Territorial Cooperation 2007–2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–2013; the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2013; and the MIUR-PRIN, contract number 2008P8BLNF (2008). R. Di Fiore was a recipient of a fellowship granted by MIUR (contract number 867/06/07/2011); D. Carlisi was a recipient of a fellowship granted by MIUR (contract number 2223/12/19/2011); R. Drago-Ferrante was a recipient of a fellowship granted by MIUR-PRIN (contract number 144/01/26/2012); M. Marcatti and F. Querques are PhD students supported by Italian Ministry of Education, University and Research (MIUR).
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - 3AB-OS cells
KW - Cancer cell dedifferentiation
KW - Cancer stem cells
KW - Human osteosarcoma
KW - Mutant p53 gain of function
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U2 - 10.1016/j.bone.2013.12.021
DO - 10.1016/j.bone.2013.12.021
M3 - Article
C2 - 24373920
AN - SCOPUS:84891899023
SN - 8756-3282
VL - 60
SP - 198
EP - 212
JO - Bone
JF - Bone
ER -