J Breast Cancer.  2014 Sep;17(3):207-218. 10.4048/jbc.2014.17.3.207.

The Role and Regulatory Mechanism of 14-3-3 Sigma in Human Breast Cancer

• Ko S
• Kim JY
• Jeong J
• Lee JE
• Yang WI
• Jung WH

• KMID: 2286347
• DOI: http://doi.org/10.4048/jbc.2014.17.3.207

Abstract

PURPOSE
14-3-3 sigma (sigma) is considered to be an important tumor suppressor and decreased expression of the same has been reported in many malignant tumors by hypermethylation at its promoter or ubiquitin-mediated proteolysis by estrogen-responsive ring finger protein (Efp). In this study, we investigated the significance of 14-3-3 sigma expression in human breast cancer and its regulatory mechanism.
METHODS
Efp was silenced using small interfering RNA (siRNA) in the MCF-7 breast cancer cell line in order to examine its influence on the level of 14-3-3 sigma protein. The methylation status of the 14-3-3 sigma promoter was also evaluated by methylation-specific polymerase chain reaction (PCR). The expression of Efp and 14-3-3 sigma in 220 human breast carcinoma tissues was assessed by immunohistochemistry. Other clinicopathological parameters were also evaluated.
RESULTS
Silencing Efp in the MCF-7 breast cancer cell line resulted in increased expression of 14-3-3 sigma. The Efp-positive human breast cancers were more frequently 14-3-3 sigma-negative (60.5% vs. 39.5%). Hypermethylation of 14-3-3 sigma was common (64.9%) and had an inverse association with 14-3-3 sigma positivity (p=0.072). Positive 14-3-3 sigma expression was significantly correlated with poor prognosis: disease-free survival (p=0.008) and disease-specific survival (p=0.009).
CONCLUSION
Our data suggests that in human breast cancer, the regulation of 14-3-3 sigma may involve two mechanisms: ubiquitin-mediated proteolysis by Efp and downregulation by hypermethylation. However, the inactivation of 14-3-3 sigma is probably achieved mainly by hypermethylation. Interestingly, 14-3-3 sigma turned out to be a very significant poor prognostic indicator, which is in contrast to its previously known function as a tumor suppressor, suggesting a different role of 14-3-3 sigma in breast cancer.

Keyword

Breast neoplasms; Estrogen-responsive finger protein; Methylation; SFN protein

MeSH Terms

Breast
Breast Neoplasms*
Cell Line
Disease-Free Survival
Down-Regulation
Fingers
Humans
Immunohistochemistry
Methylation
Polymerase Chain Reaction
Prognosis
Proteolysis
RNA, Small Interfering
RNA, Small Interfering

Figure

• 

  Figure 1 The increased expression of 14-3-3 σ by knocks down of estrogen-responsive ring finger protein (Efp) using small interfering RNA (siRNA). siEfp was transfected in human MCF-7 breast cancer cells. After transfection, total RNA and whole cell lysates were prepared and subjected to reverse transcription polymerase chain reaction (A) and Western blot (C). Relative levels of mRNA and protein expression were determined by densitometric scanning of the bands (B, D). The siEfp transfection resulted in increased levels of 14-3-3 σ mRNA and protein. *Corresponds to p<0.05.

• 

  Figure 2 Expression of estrogen-responsive ring finger protein (Efp) and 14-3-3 σ in breast cancer. An estrogen receptor-positive breast cancer was positive to Efp (A) and negative to 14-3-3 σ (B) while a triple-negative breast cancer was negative to Efp (C) and positive to 14-3-3 σ (D) (NovaRed with Hematoxylin counterstain, ×400).

• 

  Figure 3 Methylation-specific polymerase chain reaction (MSP) in breast cancer. Methylated and unmethylated bands on MSP. Methylated bands which are brighter than the corresponding unmethylated band or the ones as dense as the universally methylated DNA control were determined as hyper-methylated. 8, 18, 74, 78, and 92=case numbers; MCF=MCF-7 cell line; PC=universally methylated DNA; U=unmethylated band; M=methylated band.

• 

  Figure 4 Disease-free survival and disease-specific survival curves according to 14-3-3 σ expression in each subgroup. (A, C, E) Disease-free survival; (B, D, F) Disease-specific survival. (A, B) Total cases: 14-3-3 σ expression was significantly associated with an increased risk of recurrence and disease-related death. (C, D) Receptor-positive group: 14-3-3 σ expression was associated with an increased risk of recurrence and disease-related death, but statistically insignificant. (E, F) Triple-negative group: 14-3-3 σ expression was not associated with an increased risk of recurrence. But 14-3-3 σ expression was significantly associated with an increased risk of disease-related death. Dashed line, 14-3-3 σ negative; linear line, 14-3-3 σ positive.

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