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PIN3 duplication may be partially responsible for TP53 haploinsufficiency
Marta Winiecka-Klimek1, Malgorzata Szybka, Piotr Rieske
1Department of Tumor Biology, Medical University of Lodz, Zeligowskiego 7/9, 90-752, Lodz, Poland. marta.winiecka@gmail.com.
Background:
Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon.
Methods:
250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed.
Results:
Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes.
Conclusion:
TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.
Insights
A specific gene variant (PIN3 A2) in TP53 influences gene expression, potentially causing cancer through haploinsufficiency. This finding highlights the importance of TP53 allele-specific expression in cancer development.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Cancer cells may silence wild-type TP53, degrade its mRNA, or overproduce mutated TP53 mRNA.
- Sequencing revealed discrepancies between TP53 DNA and cDNA, suggesting allele-specific expression differences.
- The polymorphism within intron 3 (PIN3) was hypothesized to cause unequal TP53 gene expression levels between alleles.
Purpose of the Study:
- To investigate if PIN3 polymorphic variants (A1 or A2) in heterozygotes are linked to higher TP53 expression.
- To determine if this differential expression contributes to TP53 haploinsufficiency.
- To explore the role of PIN3 in TP53 mRNA expression regulation.
Main Methods:
- Analysis of 250 tumor samples.
- Bacterial subcloning and sequencing to assess PIN3 variant involvement in TP53 mRNA expression.
- Dual luciferase reporter assays to quantify allelic expression.
- Bioinformatic analysis to identify DNA structural influences.
Main Results:
- Haplotype analysis indicated mutated TP53 templates predominated in cDNA sequencing for samples with heterozygous TP53 mutations and PIN3 heterozygosity.
- Of 30 samples with TP53 mutations and biased allelic expression, 6 were heterozygous for PIN3 (A1/A2), all with mutations in the longer allele (A2) (p = 0.04).
- Reporter assays showed significantly higher luciferase activity for the A2 construct compared to A1 (A2/A1 ratio 1.16-1.59, p < 0.019).
- Bioinformatic analysis revealed PIN3 duplication stabilizes G-quadruplexes.
Conclusions:
- TP53 alleles are not equivalent in regulating TP53 mRNA expression.
- In PIN3-heterozygous individuals, a single TP53 mutation in the longer allele may be sufficient to impair function.
- Secondary DNA structures like quadruplexes may contribute to PIN3-dependent TP53 haploinsufficiency.
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