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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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Knockdown of p53 in p53R172H HCC cell lines results in increased expression of several p53 family transcriptional targets.A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53R172H. Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53fl/fl and p53R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.
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pone.0123816.g005: Knockdown of p53 in p53R172H HCC cell lines results in increased expression of several p53 family transcriptional targets.A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53R172H. Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53fl/fl and p53R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.

Mentions: We therefore evaluated a subset of the target genes analyzed in Fig 4, covering multiple cellular processes, to determine whether their mRNA levels increased upon knockdown of p53R172H. p53 knockdown was performed in two additional p53R172H-derived cell lines and knockdown confirmed by immunoblot (Fig 5A). When added to the 2 knockdown lines previously characterized (Fig 3), this generated a panel of 4 knockdown cell lines, and paired controls, for analysis. We found that the levels of p21, Cldn1, and Rad51 were commonly increased in knockdown cells relative to paired control cells (Fig 5B). This suggests that mutant p53 suppressed their expression, potentially by functionally interfering with p63 and p73.


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

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

Knockdown of p53 in p53R172H HCC cell lines results in increased expression of several p53 family transcriptional targets.A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53R172H. Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53fl/fl and p53R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123816.g005: Knockdown of p53 in p53R172H HCC cell lines results in increased expression of several p53 family transcriptional targets.A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53R172H. Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53fl/fl and p53R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.
Mentions: We therefore evaluated a subset of the target genes analyzed in Fig 4, covering multiple cellular processes, to determine whether their mRNA levels increased upon knockdown of p53R172H. p53 knockdown was performed in two additional p53R172H-derived cell lines and knockdown confirmed by immunoblot (Fig 5A). When added to the 2 knockdown lines previously characterized (Fig 3), this generated a panel of 4 knockdown cell lines, and paired controls, for analysis. We found that the levels of p21, Cldn1, and Rad51 were commonly increased in knockdown cells relative to paired control cells (Fig 5B). This suggests that mutant p53 suppressed their expression, potentially by functionally interfering with p63 and p73.

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