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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 is required for transformation-related phenotypes in HCC cell lines.A) Immunoblot of p53 depicting 3 HCC cell lines infected with shRNA as compared to an empty vector control labeled pGIPZ. B) Transwell migration assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test, are 0.020 and 0.022 for R172H lines 1 and 2, respectively. The number of migrating cells in each pGIPZ control infection was set to 100%. C) Soft agar colony formation assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test are 0.044 and 0.004 for R172H lines 1 and 2, respectively. The number of colonies for each pGIPZ control infection was set to 100%.
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pone.0123816.g003: p53R172H is required for transformation-related phenotypes in HCC cell lines.A) Immunoblot of p53 depicting 3 HCC cell lines infected with shRNA as compared to an empty vector control labeled pGIPZ. B) Transwell migration assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test, are 0.020 and 0.022 for R172H lines 1 and 2, respectively. The number of migrating cells in each pGIPZ control infection was set to 100%. C) Soft agar colony formation assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test are 0.044 and 0.004 for R172H lines 1 and 2, respectively. The number of colonies for each pGIPZ control infection was set to 100%.

Mentions: In previous studies analyzing the gain-of-function properties of mutant p53 proteins, a hallmark experiment was the demonstration that knockdown of mutant p53 abrogated the transformed phenotype [20,22]. We therefore determined whether depletion of mutant p53 similarly impacted our HCC cell lines. We knocked down p53 in 2 cell lines derived from p53R172H tumors and knockdown confirmed by immunoblotting (Fig 3A). As a control for potential off-target effects, we introduced the p53-targeting shRNA into a p53 cell line. We observed that p53 knockdown impaired cell migration (Fig 3B) and soft agar colony formation (Fig 3C) relative to controls in cells expressing the R172H mutant, suggesting that mutant p53 is required for these phenotypes in p53R172H-expressing HCC cell lines. Importantly, expression of the p53-targeting shRNA in a p53 cell line did not impact cell migration or soft agar colony formation, confirming that these effects result from suppression of mutant p53 and not off-target effects.


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

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

p53R172H is required for transformation-related phenotypes in HCC cell lines.A) Immunoblot of p53 depicting 3 HCC cell lines infected with shRNA as compared to an empty vector control labeled pGIPZ. B) Transwell migration assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test, are 0.020 and 0.022 for R172H lines 1 and 2, respectively. The number of migrating cells in each pGIPZ control infection was set to 100%. C) Soft agar colony formation assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test are 0.044 and 0.004 for R172H lines 1 and 2, respectively. The number of colonies for each pGIPZ control infection was set to 100%.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123816.g003: p53R172H is required for transformation-related phenotypes in HCC cell lines.A) Immunoblot of p53 depicting 3 HCC cell lines infected with shRNA as compared to an empty vector control labeled pGIPZ. B) Transwell migration assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test, are 0.020 and 0.022 for R172H lines 1 and 2, respectively. The number of migrating cells in each pGIPZ control infection was set to 100%. C) Soft agar colony formation assay of p53 shRNA-infected HCC cell lines. A p53- cell line was used as an experimental control. p-values, calculated by Student’s t-test are 0.044 and 0.004 for R172H lines 1 and 2, respectively. The number of colonies for each pGIPZ control infection was set to 100%.
Mentions: In previous studies analyzing the gain-of-function properties of mutant p53 proteins, a hallmark experiment was the demonstration that knockdown of mutant p53 abrogated the transformed phenotype [20,22]. We therefore determined whether depletion of mutant p53 similarly impacted our HCC cell lines. We knocked down p53 in 2 cell lines derived from p53R172H tumors and knockdown confirmed by immunoblotting (Fig 3A). As a control for potential off-target effects, we introduced the p53-targeting shRNA into a p53 cell line. We observed that p53 knockdown impaired cell migration (Fig 3B) and soft agar colony formation (Fig 3C) relative to controls in cells expressing the R172H mutant, suggesting that mutant p53 is required for these phenotypes in p53R172H-expressing HCC cell lines. Importantly, expression of the p53-targeting shRNA in a p53 cell line did not impact cell migration or soft agar colony formation, confirming that these effects result from suppression of mutant p53 and not off-target effects.

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