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14-3-3σ down-modulation: a ubiquitous marker for breast cancer?
Breast Cancer Research volume 2, Article number: 66683 (2000)
A number of promising molecular markers of breast tumor progression have been recently elucidated, most of which are altered in only a subset of breast cancers. Previous reports identified 14-3-3σ (σ), a molecule normally activated at the G2 cell cycle checkpoint in response to DNA damaging agents, as a putative molecular marker that is downregulated in breast tumor cells. Because these initial studies were performed in only a few breast carcinoma cell lines, it was not clear to what extent σ serves as a general marker of breast tumor progression.
To monitor the expression patterns of σ in an extensive panel of breast epithelial cell lines, both nonmalignant and tumorigenic, and in primary breast tumors. To explore the molecular mechanism of σ gene silencing and to examine how σ downmodulation contributes to tumor progression in the breast.
Aberrant promoter methylation is proving to be a critical epigenetic mechanism contributing to tumorigenic progression. This report provides compelling evidence that the DNA damage response gene 14-3-3σ is inactivated in breast tumors by a methylation-dependent gene silencing mechanism and that this negative regulation may be causal to tumorigenic progression. While the data indicate that 14-3-3σ is the consistent invasive breast cancer marker identified to date, it is not yet clear whether this protein will serve as a useful indicator for early detection.
Northern blots were performed to evaluate the expression of σ in nontumorigenic human mammary epithelial cells (both primary and immortalized), in two breast carcinoma cell lines and 48 primary breast tumors (invasive ductal carcinomas). Genetic causes of downmodulation were explored in σ-negative samples using PCR-based assays of loss of heterozygosity (LOH) and by direct gene sequencing. Methylation status of σ promoter sequences were evaluated in both malignant and nonmalignant cell types using sodium bisulfite DNA sequencing and methylation-specific PCR. Finally, the efficacy of DNA damage repair at G1- and G2-specific checkpoints was assayed by scoring the frequency of asymmetric chromosomal segregation during metaphase in both σ-positive and σ-negative cells exposed to stage-specific ionizing radiation.
The expression of σ, while clearly detectable in nonmalignant human mammary epithelial cells (ie six non-immortalized strains and five immortalized strains), was found to be absent in three breast carcinoma cell lines and in 45 of 48 (94%) primary breast tumors. Examination of 45 paired sets of normal and tumor patient DNAs showed only one LOH. Direct sequence of sigma coding region cDNAs from σ-negative cells (two cell lines and seven tumor tissues) showed no destabilizing genetic mutations that could account for the reduced expression levels. A striking correlation, however, was demonstrated between σ downmodulation and σ promoter hypermethylation: four σ-positive cell lines displayed unmethylated σ promoter CpG island sequences, and σ-negative cells (two cell lines and 10 primary breast tumors) showed complete or partial methylation of the σCpG island. Moreover, treatment of two σ-negative breast carcinoma cell lines with the DNA methyl transferase inhibitor, 5-aza-dC, led to demethylation of the σCpG region and reactivation of σ gene expression. Finally, while σ-positive and σ-negative cells lines both displayed similar G1 cell cycle checkpoint control responses, they exhibited different G2 checkpoint functions with σ-negative cells showing up to two-fold more G2-type chromosomal aberrations than observed with σ-positive cells.
The demonstration that 14-3-3σ is reproducibly downmodulated in a large selection of breast carcinoma cell lines and tumor samples suggests that it may be a good candidate for a general marker for invasive breast carcinoma. Loss of σ expression appears not to be due to genetic changes such as LOH or more subtle sequence mutations, but rather is regulated.
Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR, Sukumar S: High frequency of hypermethylation at the 14-3-3σ locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA. 2000, 97: 6049-6054.
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Schmeichel, K. 14-3-3σ down-modulation: a ubiquitous marker for breast cancer?. Breast Cancer Res 2, 66683 (2000). https://doi.org/10.1186/bcr-2000-66683