Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells

Riccardo Di Fiore, Michela Marcatti, Rosa Drago-Ferrante, Antonella D'Anneo, Michela Giuliano, Daniela Carlisi, Anna De Blasio, Francesca Querques, Lucio Pastore, Giovanni Tesoriere, Renza Vento

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)198-212
Number of pages15
JournalBone
Volume60
DOIs
StatePublished - Mar 2014
Externally publishedYes

Fingerprint

Neoplastic Stem Cells
Osteosarcoma
p53 Genes
Mutant Proteins
Neoplasms
Cell Dedifferentiation
Apoptosis
Polymerase Chain Reaction
Growth
Tumor Suppressor Genes
Fluorescence In Situ Hybridization
Methylation
Small Interfering RNA
Fluorescent Antibody Technique
Cell Cycle
Up-Regulation
Western Blotting
Drug Therapy
Cell Line
Survival

Keywords

  • 3AB-OS cells
  • Cancer cell dedifferentiation
  • Cancer stem cells
  • Human osteosarcoma
  • Mutant p53 gain of function

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Histology
  • Physiology

Cite this

Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells. / Di Fiore, Riccardo; Marcatti, Michela; Drago-Ferrante, Rosa; D'Anneo, Antonella; Giuliano, Michela; Carlisi, Daniela; De Blasio, Anna; Querques, Francesca; Pastore, Lucio; Tesoriere, Giovanni; Vento, Renza.

In: Bone, Vol. 60, 03.2014, p. 198-212.

Research output: Contribution to journalArticle

Di Fiore, R, Marcatti, M, Drago-Ferrante, R, D'Anneo, A, Giuliano, M, Carlisi, D, De Blasio, A, Querques, F, Pastore, L, Tesoriere, G & Vento, R 2014, 'Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells', Bone, vol. 60, pp. 198-212. https://doi.org/10.1016/j.bone.2013.12.021
Di Fiore, Riccardo ; Marcatti, Michela ; Drago-Ferrante, Rosa ; D'Anneo, Antonella ; Giuliano, Michela ; Carlisi, Daniela ; De Blasio, Anna ; Querques, Francesca ; Pastore, Lucio ; Tesoriere, Giovanni ; Vento, Renza. / Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells. In: Bone. 2014 ; Vol. 60. pp. 198-212.
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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.",
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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

PY - 2014/3

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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.

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KW - Mutant p53 gain of function

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