J Breast Cancer.  2019 Jun;22(2):172-184. 10.4048/jbc.2019.22.e21.

Overexpression of Tumor Protein p53-regulated Apoptosis-inducing Protein 1 Regulates Proliferation and Apoptosis of Breast Cancer Cells through the PI3K/Akt Pathway

• Liang Y
• Wang S
• Liu J

• KMID: 2450113
• DOI: http://doi.org/10.4048/jbc.2019.22.e21

Abstract

PURPOSE
Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) functions in various cancers. We studied the effect and molecular mechanism of TP53AIP1 in breast cancer.
METHODS
The degree of correlation between TP53AIP1 expression and overall survival in patients with breast cancer was obtained from the online The Cancer Genome Atlas database. Six of the TP53AIP1 levels in the tumor and adjacent non-tumor tissues randomly selected from 38 breast cancer patients were determined. Transgenic technology was used to enhance the expression of TP53AIP1 in breast cancer cell lines, MDA-MB-415 and MDA-MB-468, and to observe the effects of gene overexpression on the proliferation, cell cycle, and apoptosis of breast cancer cells. The molecular mechanism of association between cell cycle- and apoptosis-related factors and the phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) pathway was also studied.
RESULTS
The messenger RNA and protein expression levels of TP53AIP1 in cancer tissues were significantly lower than those in the control group. TP53AIP1 overexpression inhibits cell viability. The mechanism of TP53AIP1 inhibition of proliferation and growth of breast cancer cells includes cell cycle arrest, apoptosis promotion (p < 0.01), promotion of the expression of cleaved-caspase-3 (p < 0.01), cleaved-caspase-9 (p < 0.01), B cell lymphoma/leukemia-2 (Bcl-2)-associated X protein, and p53 (p < 0.01), and the inhibition of Bcl-2, Ki67, and PI3K/Akt pathways (p < 0.01).
CONCLUSION
TP53AIP1 may be a novel tumor suppressor gene in breast cancer and can potentially be used as an effective target gene for the treatment of breast cancer.

Keyword

Apoptosis; Breast neoplasms; Cell proliferation; Genes, p53

MeSH Terms

Apoptosis*
Breast Neoplasms*
Breast*
Cell Cycle Checkpoints
Cell Line
Cell Proliferation
Cell Survival
Genes, p53
Genes, Tumor Suppressor
Genome
Humans
Phosphotransferases
RNA, Messenger
Phosphotransferases
RNA, Messenger

Figure

• 

  Figure 1 TP53AIP1 is down-regulated in patients with breast cancer. (A) Data from The Cancer Genome Atlas database were collected, and the data showed that breast cancer patients with low level of TP53AIP1 had lower survival rates. (B) Quantitative real-time polymerase chain reaction assay verified down-regulated overall level of TP53AIP1 mRNA in breast cancer patients. (C) Western blot showed down-regulated protein levels of TP53AIP1 in most breast cancer patients, and the representative ones are displayed. (D) Immunohistochemistry assay showed negative protein expression of TP53AIP1 in invasive breast cancer tissues and positive brown TP53AIP1 protein expression in adjacent tissues under 100- and 200-fold magnifications. TP53AIP1 = tumor protein p53-regulated apoptosis-inducing protein 1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; mRNA = messenger RNA; NS = not significant. *p < 0.05 and †p < 0.01 vs. adjacent tissue.

• 

  Figure 2 Overexpression of TP53AIP1 inhibits viability of breast cancer cells. RT-qPCR (A) and Western blot (B) were used to show increased mRNA and protein levels of TP53AIP1 in TP53AIP1 (MDA-MB-415) group. RT-qPCR (C) and western blot (D) were used to show increased mRNA and protein levels of TP53AIP1 in TP53AIP1 (MDA-MB-468) group. Cell Counting Kit-8 assay was used to show inhibited cell viabilities in TP53AIP1 (MDA-MB-415) group (E) and TP53AIP1 (MDA-MB-468) group (F). TP53AIP1 = tumor protein p53-regulated apoptosis-inducing protein 1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; mRNA = messenger RNA; RT-qPCR = real-time-quantitative polymerase chain reaction; NS = not significant; OD = optical density; NC = negative control. *p < 0.01 vs. NC-vector group.

• 

  Figure 3 Overexpression of TP53AIP1 promotes cell cycle arrest and apoptosis of breast cancer cells. Cell cycle arrest (A) and apoptosis rates (B) were found to be promoted in TP53AIP1 (MDA-MB-415) group. Cell cycle arrest (C) and apoptosis rates (D) were found promoted in TP53AIP1 (MDA-MB-415) group and (MDA-MB-468) group. The rates were determined using flow cytometry. TP53AIP1 = tumor protein p53-regulated apoptosis-inducing protein 1; NC = negative control; FL2 = fidgetin-like 2; NS = not significant. *p < 0.01 vs. NC-vector group.

• 

  Figure 4 Overexpression of TP53AIP1 promotes cell cycle arrest and apoptosis of breast cancer cells by regulating cell cycle and apoptosis related factors. The mRNA levels of Bax and p53 increased, and Bcl-2 and Ki67 levels decreased in TP53AIP1 (MDA-MB-415) (A-D) and TP53AIP1 (MDA-MB-468) (F-I) groups. The protein levels of cleaved-caspase-3, cleaved-caspase-9, Bax and p53 increased and Bcl-2 and Ki67 levels decreased in TP53AIP1 (MDA-MB-415) (E) and TP53AIP1 (MDA-MB-468) (J) groups. TP53AIP1 = tumor protein p53-regulated apoptosis-inducing protein 1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; Bcl-2 = B cell lymphoma/leukemia-2; Bax = Bcl-2-associated X protein; mRNA = messenger RNA; NC = negative control; NS = not significant. *p < 0.05 and †p < 0.01 vs. NC-vector group.

• 

  Figure 5 Overexpression of TP53AIP1 promotes cell cycle arrest and apoptosis of breast cancer cells via inhibiting the PI3K/Akt pathway. The percentage of p-PI3K/PI3K, p-Akt/Akt, and Mdm2 levels were suppressed in TP53AIP1 (MDA-MB-415) (A) and TP53AIP1 (MDA-MB-468) (B) groups. TP53AIP1 = tumor protein p53-regulated apoptosis-inducing protein 1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; NC = negative control; NS = not significant; PI3K = phosphoinositide 3-kinases; Akt = protein kinase B; p-Akt = phosphorylated-Akt; p-PI3K = phosphorylated-PI3K; Mdm2 = mouse double minute 2 homolog. *p < 0.01 vs. NC-vector group.

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