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The p53R172H mutant does not enhance hepatocellular carcinoma development and progression.

Ahronian LG, Driscoll DR, Klimstra DS, Lewis BC - PLoS ONE (2015)

Bottom Line: However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth.These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 cells, resulting in equivalent phenotypes.Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates "prometastatic" gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

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p53R172H does not result in gain-of-function properties in an HCC mouse model.A) Kaplan-Meier curve displaying tumor free survival of p53fl/fl and p53R172H littermates. Mice were assessed over a period of 9 months and sacrificed when illness was apparent. Only mice whose death was perceived to be due to HCC were included, otherwise the mouse was censored. Censored animals are represented in the figure as tick marks on the day of death. B) Total tumor burden in the survival cohort. Individual tumor volume was calculated using the formula for an ellipsoid. All of the tumor volumes for a given mouse were summed to arrive at the tumor burden per animal. C) H&E staining demonstrating commonly seen architecture in HCC mouse model. D) Number of p53R172H and p53fl/fl mice with (grey) or without (black) grossly visible metastatic lesions.
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pone.0123816.g001: p53R172H does not result in gain-of-function properties in an HCC mouse model.A) Kaplan-Meier curve displaying tumor free survival of p53fl/fl and p53R172H littermates. Mice were assessed over a period of 9 months and sacrificed when illness was apparent. Only mice whose death was perceived to be due to HCC were included, otherwise the mouse was censored. Censored animals are represented in the figure as tick marks on the day of death. B) Total tumor burden in the survival cohort. Individual tumor volume was calculated using the formula for an ellipsoid. All of the tumor volumes for a given mouse were summed to arrive at the tumor burden per animal. C) H&E staining demonstrating commonly seen architecture in HCC mouse model. D) Number of p53R172H and p53fl/fl mice with (grey) or without (black) grossly visible metastatic lesions.

Mentions: To determine if p53R172H exhibits gain-of-function properties in HCC, we generated Albumin-tva, LSL-Trp53R172H/flox, Albumin-cre mice (hereafter referred to as p53R172H mice) and Albumin-tva, Trp53flox/flox, Albumin-cre (p53fl/fl) littermates. These mice were injected with DF1 cells producing RCAS-PyMT to induce HCC, as done previously [11,37]. In this model, loss of p53 function promotes metastasis, which can be enhanced through concomitant deletion of Ink4a/Arf [11,37]. Therefore, if the p53R172H mutant has gain-of-function properties, these should be observed in this model as enhanced tumor progression. We found that the presence of the mutated p53 allele did not impact tumor-free survival (Fig 1A). Immunohistochemical staining of tumor specimens detected nuclear p53 in p53R172H tumors but not p53fl/fl tumors, demonstrating specific expression within the p53 mutant tumors (S1 Fig). Analysis of genomic DNA isolated from a subset of liver tumors also confirmed recombination of the LSL cassette in the p53R172H tumors confirming activation of the mutant p53 allele (S1 Fig).


The p53R172H mutant does not enhance hepatocellular carcinoma development and progression.

Ahronian LG, Driscoll DR, Klimstra DS, Lewis BC - PLoS ONE (2015)

p53R172H does not result in gain-of-function properties in an HCC mouse model.A) Kaplan-Meier curve displaying tumor free survival of p53fl/fl and p53R172H littermates. Mice were assessed over a period of 9 months and sacrificed when illness was apparent. Only mice whose death was perceived to be due to HCC were included, otherwise the mouse was censored. Censored animals are represented in the figure as tick marks on the day of death. B) Total tumor burden in the survival cohort. Individual tumor volume was calculated using the formula for an ellipsoid. All of the tumor volumes for a given mouse were summed to arrive at the tumor burden per animal. C) H&E staining demonstrating commonly seen architecture in HCC mouse model. D) Number of p53R172H and p53fl/fl mice with (grey) or without (black) grossly visible metastatic lesions.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401698&req=5

pone.0123816.g001: p53R172H does not result in gain-of-function properties in an HCC mouse model.A) Kaplan-Meier curve displaying tumor free survival of p53fl/fl and p53R172H littermates. Mice were assessed over a period of 9 months and sacrificed when illness was apparent. Only mice whose death was perceived to be due to HCC were included, otherwise the mouse was censored. Censored animals are represented in the figure as tick marks on the day of death. B) Total tumor burden in the survival cohort. Individual tumor volume was calculated using the formula for an ellipsoid. All of the tumor volumes for a given mouse were summed to arrive at the tumor burden per animal. C) H&E staining demonstrating commonly seen architecture in HCC mouse model. D) Number of p53R172H and p53fl/fl mice with (grey) or without (black) grossly visible metastatic lesions.
Mentions: To determine if p53R172H exhibits gain-of-function properties in HCC, we generated Albumin-tva, LSL-Trp53R172H/flox, Albumin-cre mice (hereafter referred to as p53R172H mice) and Albumin-tva, Trp53flox/flox, Albumin-cre (p53fl/fl) littermates. These mice were injected with DF1 cells producing RCAS-PyMT to induce HCC, as done previously [11,37]. In this model, loss of p53 function promotes metastasis, which can be enhanced through concomitant deletion of Ink4a/Arf [11,37]. Therefore, if the p53R172H mutant has gain-of-function properties, these should be observed in this model as enhanced tumor progression. We found that the presence of the mutated p53 allele did not impact tumor-free survival (Fig 1A). Immunohistochemical staining of tumor specimens detected nuclear p53 in p53R172H tumors but not p53fl/fl tumors, demonstrating specific expression within the p53 mutant tumors (S1 Fig). Analysis of genomic DNA isolated from a subset of liver tumors also confirmed recombination of the LSL cassette in the p53R172H tumors confirming activation of the mutant p53 allele (S1 Fig).

Bottom Line: However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth.These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 cells, resulting in equivalent phenotypes.Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates "prometastatic" gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

Show MeSH
Related in: MedlinePlus