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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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Related in: MedlinePlus

p53R172H and p53fl/fl HCC cell lines do not display differential expression of p53 family transcriptional targets.A) qRT-PCR of DNA damage-related targets of p53 family transcription factors. B) qRT-PCR analysis of p53 family targets related to cell signaling. C) qRT-PCR analysis of p53 family targets related to other cell processes. Dark circles indicate p53fl/fl cell lines, while open circles are p53R172H cell lines.
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pone.0123816.g004: p53R172H and p53fl/fl HCC cell lines do not display differential expression of p53 family transcriptional targets.A) qRT-PCR of DNA damage-related targets of p53 family transcription factors. B) qRT-PCR analysis of p53 family targets related to cell signaling. C) qRT-PCR analysis of p53 family targets related to other cell processes. Dark circles indicate p53fl/fl cell lines, while open circles are p53R172H cell lines.

Mentions: The above data suggest that while p53R172H is required for transformation-associated phenotypes in HCC cells expressing the mutant, the presence of mutant p53 does not enhance these properties beyond what is observed in p53 cell lines. This suggests, potentially, that similar pathways are impacted in both contexts. Since p53R172H is known to bind to, and inhibit, other p53 family transcription factors as a mechanism of its gain-of-function properties in other cancer types, we examined the mRNA levels of a collection of genes known to be regulated by the related p63 and p73 proteins. Previously published studies have described several genes regulated by p63 and p73. We curated a collection of published target genes from such studies [38–40], and selected a subset of these genes that are involved in a range of cellular processes for characterization in our HCC cell lines. We found that the mRNA levels of p53 family target genes involved in cell signaling (Fig 4A), DNA damage (Fig 4B), and cellular processes (Fig 4C), are unchanged between p53R172H and p53fl/fl cell lines.


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

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

p53R172H and p53fl/fl HCC cell lines do not display differential expression of p53 family transcriptional targets.A) qRT-PCR of DNA damage-related targets of p53 family transcription factors. B) qRT-PCR analysis of p53 family targets related to cell signaling. C) qRT-PCR analysis of p53 family targets related to other cell processes. Dark circles indicate p53fl/fl cell lines, while open circles are p53R172H cell lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123816.g004: p53R172H and p53fl/fl HCC cell lines do not display differential expression of p53 family transcriptional targets.A) qRT-PCR of DNA damage-related targets of p53 family transcription factors. B) qRT-PCR analysis of p53 family targets related to cell signaling. C) qRT-PCR analysis of p53 family targets related to other cell processes. Dark circles indicate p53fl/fl cell lines, while open circles are p53R172H cell lines.
Mentions: The above data suggest that while p53R172H is required for transformation-associated phenotypes in HCC cells expressing the mutant, the presence of mutant p53 does not enhance these properties beyond what is observed in p53 cell lines. This suggests, potentially, that similar pathways are impacted in both contexts. Since p53R172H is known to bind to, and inhibit, other p53 family transcription factors as a mechanism of its gain-of-function properties in other cancer types, we examined the mRNA levels of a collection of genes known to be regulated by the related p63 and p73 proteins. Previously published studies have described several genes regulated by p63 and p73. We curated a collection of published target genes from such studies [38–40], and selected a subset of these genes that are involved in a range of cellular processes for characterization in our HCC cell lines. We found that the mRNA levels of p53 family target genes involved in cell signaling (Fig 4A), DNA damage (Fig 4B), and cellular processes (Fig 4C), are unchanged between p53R172H and p53fl/fl cell lines.

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