J Breast Cancer.  2019 Mar;22(1):15-28. 10.4048/jbc.2019.22.e11.

Pokemon Inhibits Transforming Growth Factor β-Smad4-Related Cell Proliferation Arrest in Breast Cancer through Specificity Protein 1

• Chen L
• Zhong J
• Liu JH
• Liao DF
• Shen YY
• Zhong XL
• Xiao X
• Ding WJ
• Peng XD
• Xiong W
• Zu XY

• KMID: 2441848
• DOI: http://doi.org/10.4048/jbc.2019.22.e11

Abstract

PURPOSE
Pokemon, also known as ZBTB7A, belongs to the POZ and Krüppel (POK) family of transcription repressors and is implicated in tumor progression as a key proto-oncogene. This present study aimed at determining the mechanism by which Pokemon inhibits transforming growth factor β (TGFβ)-Smad4 pathway-dependent proliferation arrest of breast cancer cells via specificity protein 1 (SP1).
METHODS
Over-expressing plasmid or small interfering RNA (siRNA) transfection was used to regulate Pokemon levels. The EdU incorporation assay, MTS assay, and clone formation were used to identify the inhibitory effect of Pokemon siRNA on cell proliferation. Quantitative real-time polymerase chain reaction assay confirmed that Pokemon deletion inhibited the expression of proliferation-associated genes. The dual-luciferase reporter assay, electrophoretic mobility shift assay, and co-immunoprecipitation assay were used to analyze binding between Pokemon, Smad4, and SP1.
RESULTS
Pokemon deletion induced proliferation arrest of breast cancer cells and inhibited the expression of proliferation-associated genes, especially Smad4. Pokemon bound with SP1 to interdict Smad4 promoter activity. Information on clinical samples was obtained from The Cancer Genome Atlas data, in which the Pokemon mRNA levels showed a negative correlation with Smad4 levels in different subtypes of breast cancer in two independent datasets.
CONCLUSION
We demonstrated that Pokemon binds to SP1 to down-regulate Smad4 expression, thereby promoting proliferation of breast cancer cells. This suggests that Pokemon is a potential TGFβ-signaling participant in breast cancer progression.

Keyword

Breast neoplasms; Cell proliferation; Pokemon protein, human; Smad4 protein; SP1

MeSH Terms

Breast Neoplasms*
Breast*
Cell Proliferation*
Clone Cells
Dataset
Electrophoretic Mobility Shift Assay
Genome
Humans
Immunoprecipitation
Plasmids
Proto-Oncogenes
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Sensitivity and Specificity*
Smad4 Protein
Transfection
Transforming Growth Factors*
RNA, Messenger
RNA, Small Interfering
Smad4 Protein
Transforming Growth Factors

Figure

• 

  Figure 1 Aggravation of TGFβ-induced cell proliferation arrest after Pokemon deletion in breast cancer cells. After transiently transfected with Neg-siR, Poke-siR (100 nM) or treated with TGFβ1 (10 ng/mL) for 48 hours in MDA-MB-231 cells (A, B), the number of EdU positive cells (EdU+, green color), which represented DNA replication ability, was reduced than control group. (C-E) Alternative siRNA or TGFβ treatment for 48 hours, the viability and proliferation of breast cancer cells were significantly weakened. (F) The colony number of breast cancer cells was also decreased by the 2 treatments. In the above experiments, Pokemon deficiency could promote the growth arrest by TGFβ protein treatment (n = 4). TGFβ = transforming growth factor β; Neg-siR = negative siRNA; Poke-siR = Pokemon siRNA. *p < 0.01, †p < 0.001.

• 

  Figure 2 Negative connection between Pokemon and TGFβ-Smad4. (A) Up-regulation of Pokemon affected a series of cell signaling pathway, which was evaluated by the gene array analysis. (B) The mRNA levels of cell invasion-related genes were detected by quantitative real-time polymerase chain reaction after the transfection of Poke-OE or Con. (C) IF assay identified up- or down-regulation of Pokemon reduced or increased expression of Smad4. (D) Western blot results indicated transfection of over-expressed plasmid of Pokemon reduced protein levels of Smad4, and the Pokemon siRNA transfection showed the adverse effects (n = 4). TGFβ = transforming growth factor β; Poke-OE = Pokemon over-expression plasmid with GFP label; Con = negative plasmid; IF= immunofluorescence; MAPK= mitogen-activated protein kinase; DAPI = 4',6-diamidino-2-phenylindole. *p < 0.05, †p < 0.01.

• 

  Figure 3 Pokemon regulation on multiple proliferation associated genes. (A-C) Quantitative real-time polymerase chain reaction assay identified that Pokemon knockdown increased TGFβ1 gene level and reduced several proliferation-associated gene levels in MDA-MB-231, T47D and MCF-10A cells (n = 4). (D) Protein levels of CYCLIND1 and CDKN1A were reduced by Pokemon knockdown. TGFβ = transforming growth factor β; Neg-siR = negative siRNA; Poke-siR = Pokemon siRNA; CDKN1A = cyclin dependent kinase inhibitor 1A; CDKN1B = cyclin dependent kinase inhibitor 1B; GADD45B = growth arrest and DNA-damage-inducible, beta; IGFBP3 = insulin-like growth factor-binding protein 3; ATF3 = activating transcription factor 3; CDC6 = cell division cycle 6; EMP1 = epithelial membrane protein 1; PTK2 = protein tyrosine kinase 2; ACVR1 = activin A receptor, type I. *p < 0.05, †p < 0.01, ‡p < 0.001.

• 

  Figure 4 Indirect inhibition of Pokemon on Smad4 promoter activity through binding with SP1. (A) Dual luciferase reporter system showed that transfection of Poke-OE reduced Smad4 luciferase activity (pLuc366 and pLuc207, Smad4 expression plasmid vectors in MDB-MA-231 cells). (B) EMSA assay applied with 2 Smad4 probes proved Pokemon was not bound to promoter active regions of Smad4. (C) Co-immunoprecipitation identified that Pokemon was closely bound to SP1 protein. (D) SP1 promoted luciferase activity of Smad4, which was dose-depend weaken by Pokemon vectors (0.5 µg, 1 µg) in breast cancer cells (n = 3). SP1 = specificity protein 1; Poke-OE = Pokemon over-expressing vectors; IP = immunoprecipitation; IB = immunoblotting; Ab = antibody. *p < 0.05, †p < 0.01.

• 

  Figure 5 Higher expression level of Pokemon in luminal A type than Basal-like breast cancer tissues, which was just opposite for Smad4. (A, B) The mRNA expression of Pokemon was higher in luminal A type, but lower in basal-like type breast carcinomas, which was just opposite to Smad4. Data was obtained from 2 breast carcinoma database systems. HER2 = human epidermal growth factor receptor 2. *p < 0.05, †p < 0.01, ‡p < 0.001.

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